Protein expression systems

ABSTRACT

The present invention provides an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. In addition, the present invention provides improved recombinant protein production in host cells through the improved regulation of expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional patent applicationSer. No. 60/523,420 filed Nov. 19, 2003, entitled “Improved PseudomonasExpression Systems with Auxotrophic Selection Markers,” and U.S.Provisional patent application 60/537,147 filed Jan. 16, 2004, andentitled “Bacterial Expression Systems with Improved Repression.”

FIELD OF THE INVENTION

The present invention provides an improved expression system for theproduction of recombinant polypeptides utilizing auxotrophic selectablemarkers. In addition, the present invention provides improvedrecombinant protein production in host cells through the improvedregulation of expression.

BACKGROUND OF THE INVENTION

The use of bacterial cells to produce protein based therapeutics isincreasing in commercial importance. One of the goals in developing abacterial expression system is the production of high quality targetpolypeptides quickly, efficiently, and abundantly. An ideal host cellfor such an expression system would be able to efficiently utilize acarbon source for the production of a target polypeptide, quickly growto high cell densities in a fermentation reaction, express the targetpolypeptide only when induced, and grow on a medium that is devoid ofregulatory and environmental concerns.

There are many hurdles to the creation of a superior host cell. First,in order to produce a recombinant polypeptide, an expression vectorencoding the target protein must be inserted into the host cell. Manybacteria are capable of reverting back into an untransformed state,wherein the expression vector is eliminated from the host. Suchrevertants can decrease the fermentation efficiency of the production ofthe desired recombinant polypeptide.

Expression vectors encoding a target peptide typically include aselection marker in the vector. Often, the selection marker is a genewhose product is required for survival during the fermentation process.Host cells lacking the selection marker, such as revertants, are unableto survive. The use of selection markers during the fermentation processis intended to ensure that only bacteria containing the expressionvector survive, eliminating competition between the revertants andtransformants and reducing the efficiency of fermentation.

The most commonly used selection markers are antibiotic resistancegenes. Host cells are grown in a medium supplemented with an antibioticcapable of being degraded by the selected antibiotic resistance geneproduct. Cells that do not contain the expression vector with theantibiotic resistance gene are killed by the antibiotic. Typicalantibiotic resistance genes include tetracycline, neomycin, kanamycin,and ampicillin. The presence of antibiotic resistance genes in abacterial host cell, however, presents environmental, regulatory, andcommercial problems. For example, antibiotic resistance gene-containingproducts (and products produced by the use of antibiotic resistancegene) have been identified as potential biosafety risks forenvironmental, human, and animal health. For example, see M. Droge etal., Horizontal Gene Transfer as a Biosafety issue: A natural phenomenonof public concern, J. Biotechnology. 64(1): 75-90 (17 Sept. 1998);Gallagher, D. M., and D. P. Sinn. 1983. Penicillin-induced anaphylaxisin a patient under hypotensive anaesthesia. Oral Surg. Oral Med. OralPathol. 56:361-364; Jorro, G., C. Morales, J. V. Braso, and A. Pelaez.1996. Anaphylaxis to erythromycin. Ann. Allergy Asthma Immunol.77:456-458; F. Gebhard & K. Smalla, Transformation of Acinetobacter sp.strain BD413 by transgenic sugar beet DNA, Appl. & Environ. Microbiol.64(4):1550-54 (Apr. 1998); T. Hoffinann et al., Foreign DNA sequencesare received by a wild type strain of Aspergillus niger after co-culturewith transgenic higher plants, Curr. Genet. 27(1): 70-76 (Dec. 1994); DKMercer et al., Fate of free DNA and transformation of the oral bacteriumStreptococcus gordonoii DL1 by plasmid DNA in human saliva, Appl. &Environ. Microbiol. 65(1):6-10 (Jan 1999); R. Schubbert et al., Foreign(M13) DNA ingested by mice reaches peripheral leukocytes, spleen, andliver via the intestinal wall mucosa and can be covalently linked tomouse DNA, PNAS USA 94:961-66 (Feb. 4, 1997); and AA Salyers, Genetransfer in the mammalian intestinal tract, Curr. Opin. in Biotechnol.4(3):294-98 (Jun. 1993).

As a result of these concerns, many governmental food, drug, health, andenvironmental regulatory agencies, as well as many end users, requirethat antibiotic resistance gene nucleic acid be removed from products orbe absent from organisms for use in commerce. In addition, evidencedemonstrating clearance of the selection antibiotics from the finalproduct must be provided in order to secure regulatory clearance. TheUnited Kingdom, Canada, France, the European Community, and the UnitedStates have all addressed the use of antibiotic resistance genes infoods, animal feeds, drugs and drug production, including recombinantdrug production. Clearance of these agents, and especially demonstratingsuch clearance, is expensive, time consuming, and often only minimallyeffective.

Because of the concerns inherent in the use of antibiotic resistancegenes for selection in the production of recombinant polypeptides,alternative selection methods have been examined.

Auxotrophic Selection Markers

Auxotrophic selection markers have been utilized as an alternative toantibiotic selection in some systems. For example, auxotrophic markershave been widely utilized in yeast, due largely to the inefficiency ofantibiotic resistance selection markers in these host cells. See, forexample, JT Pronk, (2002) “Auxotrophic yeast strains in fundamental andapplied research,” App. & Envirn. Micro. 68(5): 2095-2100; Boeke et al.,(1984) “A positive selection for mutants lacking orotodine-5′-phosphatedecarboxylase activity in yeast; 5-fluoro-orotic acid resistance,” Mol.Gen. Genet. 197: 345-346; Botstein & Davis, (1982) “Principles andpractice of recombinant DNA research with yeast,” p.607-636, in J NStrathern, E W Jones. And JR Broach (ed.), The molecular biology of theyeast Saccharomyces cerevisiae, Metabolism and gene expression, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Cost & Boeke,(1996) “A useful colony color phenotype associated with the yeastselectable/counter selectable marker MET15,” Yeast 12: 939-941. However,yeast expression systems due not provide the potential speed andefficiency for producing target proteins that bacterial systems do.

Auxotrophic marker selection in bacteria has also previously beendescribed. See, for example, U.S. Pat. Nos. 4,920,048, 5,691,185,6,291,245, 6,413,768, 6,752,994, Struhl et al. (1976) PNAS USA 73;1471-1475;; MacCormick, C. A., et al., (1995) “Construction of afood-grade host/vector system for Lactococcus lactis based on thelactose operon,” FEMS Microbiol. Lett. 127:105-109; Dickely et al.(1995), “Isolation of Lactococcus lactis nonsense suppressors andconstruction of a food-grade cloning vector,” Mol. Microbiol.15:839-847; Sørensen et al., (2000) “A food-grade cloning system forindustrial strains of Lactococcus lactis,” Appl. Environ. Microbiol66:1253-1258; Fiedler & Skerra, (2001) “proBA complementation of anauxotrophic E.coli strain improves plasmid stability and expressionyield during fermenter production of a recombinant antibody fragment,”Gene 274: 111-118.

The use of auxotrophic selection markers in the previously describedcommercial scale bacterial fermentation systems has drawbacks that limittheir use. A major drawback, as noted in U.S. Pat. No. 6,413,768, isthat nutritional auxotrophic selection marker systems generally sufferfrom cross feeding. The term cross feeding refers to the ability of afirst cell, auxotrophic for a particular metabolite, to survive in theabsence of the metabolite by obtaining its supply of that metabolitefrom its environment, and typically, from the medium for which the cellis auxotrophic by utilizing excreted intermediates of the metabolite,the metabolite itself, or a prototrophic enabling molecule produced by asecond cell, prototrophic for the metabolite absent from the medium. Seealso G R Barker et al., Biochem. J. 157(1):221-27 (1976) (cross feedingof thymine in E.coli): T J Kerr & G J Tritz, J. Bact. 115(3):982-86(Sep. 1973) (cross feeding of NAD in E.coli auxotrophic for NADsynthesis); G A Sprenger et al., FEMS Microbiol. Lett. 37(3):299-304(1986) (selection of nalidixic acid to avoid the cross feeding problem).

Because cross feeding allows revertant bacteria to survive, crossfeeding decreases the overall capacity of the fermentation process toproduce the desired product at efficient and maximized levels due to thepresence of fewer target protein producing host cells.

Expression Vector Control

Another hurdle to the creation of the ideal host cell is the inefficientand low level production of target polypeptides in the fermentationprocess. Controlling expression of the target protein until optimal hostcell densities and fermentation conditions are reached allows for a moreefficient and larger yield of polypeptide. The reasons for this areseveral fold, including a more efficient utilization of a particularcarbon source and the reduction of extended metabolic stresses on thehost cell.

In many cases, however, repression of expression of the target proteinduring cell growth can be imperfect, resulting in a significant amountof expression prior to the particular induction phase. This “leaky”repression results in host cell stress, inefficient utilization ofcarbon source due to metabolic energy being diverted from normal cellgrowth to transgene, and a delay in reaching optimal cell densityinduction points, resulting in a more lengthy and costly fermentationrun, and often, a reduced yield of the target protein.

Therefore, it is an object of the present invention to provide animproved expression system for the production of target proteins,wherein the production is efficient, regulatable, and performed in amedium that minimizes of regulatory and environmental concerns.

It is another object of the present invention to provide organisms foruse as host cells in an improved expression system for the production oftarget proteins.

It is still another object of the present invention to provide processesfor the improved production of target proteins.

It is yet another object of the present invention to provide novelconstructs and nucleic acids for use in an improved expression systemfor the production of target proteins.

SUMMARY OF THE INVENTION

It has been discovered that bacterial protein production can be improvedby selecting as a host cell a Pseudomonad organism that is capable ofnon-antibiotic resistant, auxotrophic selection, and/or contains achromosomal insert of a lacI gene or derivative.

Specifically, it has been discovered that the Pseudomonad organismPseudomonas fluorescens is particularly well suited for this purpose. Tothis end, it has been surprisingly discovered that Pseudomonasfluorescens does not exhibit adverse cross feeding inhibition underauxotrophic selection during the high-cell density fermentation ofrecombinant polypeptides. Such a discovery allows for the use ofauxotrophic Pseudomonas fluorescens as host cells in the efficientproduction of high levels of recombinant polypeptides, overcoming thedrawbacks inherent with the use of antibiotic resistance selectionmarkers and the problems of auxotrophic cross feeding present in otherbacterial expression systems.

It has also been surprisingly discovered that the use of a LacI-encodinggene other than as part of a whole or truncated Plac-lacI-lacZYA operonin Pseudomonads surprisingly resulted in substantially improvedrepression of pre-induction recombinant protein expression, higher celldensities in commercial-scale fermentation, and higher yields of thedesired product in comparison with previously taught lacI-lacZYAPseudomonad chromosomal insertion (U.S. Pat. No. 5,169,760). This lacIinsertion is as effective in repressing Plac-Ptac familypromoter-controlled transgenes as a multi-copy plasmid encoding a LacIrepressor protein in Pseudomonas fluorescens, thereby eliminating theneed to maintain a separate plasmid encoding a LacI repressor protein inthe cell and reducing potential production inefficiencies caused by suchmaintenance.

It has also been discovery that the use of dual lac operator sequencesprovides superior repression of recombinant protein expression prior toinduction without a concomitant reduction in subsequent induction yieldsin Pseudomonas fluorescens

Therefore, in one aspect of the present invention, Pseudomonad organismsare provided for use as host cells in the improved production ofproteins.

In one embodiment, the Pseudomonad organisms have been geneticallymodified to induce an auxotrophy. In a particular embodiment, thePseudomonad organism is Pseudomonas fluorescens. In one embodiment, theauxotrophy is a result of genetic modifications to at least onenitrogenous base compound biosynthesis gene, or at least one amino acidbiosynthesis gene. In a further embodiment, the genetic modification isto a gene encoding an enzyme active in the uracil biosynthetic pathway,the thymidine biosynthetic pathway, or the proline biosynthetic pathway.In still a further embodiment, the genetic modification is to the pyrFgene encoding orotidine-5′-phosphate decarboxylase, the thyA geneencoding thymidylate synthase, or the proC gene encodingΔ¹-pyrroline-5-carboxylate reductase.

In another embodiment, the present invention provides Pseudomonadorganisms that have been genetically modified to provide at least onecopy of a LacI-encoding gene inserted into the genome, other than aspart of the whole or truncated Plac-lacI-lacZYA operon. In a particularembodiment, the Pseudomonad host cell is Pseudomonas fluorescens. In oneembodiment, the Pseudomonad contains a native E.coli lacI gene encodingthe LacI repressor protein. In another embodiment, the Pseudomonad cellcontains the lacI^(Q) gene. In still another embodiment, the Pseudomonadcell contains the lacI^(Q1) gene.

In another embodiment, a Pseudomonad organism is provided comprising anucleic acid construct containing a nucleic acid comprising at least onelacO sequence involved in the repression of transgene expression. In aparticular embodiment, the Pseudomonad host cell is Pseudomonasfluorescens. In one embodiment, the nucleic acid construct comprisesmore than one lacO sequence. In another embodiment, the nucleic acidconstruct comprises at least one, and preferably more than one, lacOidsequence. In one embodiment, the nucleic acid construct comprises a lacOsequence, or derivative thereof, located 3′ of a Plac family promoter,and a lacO sequence, or derivative thereof, located 5′ of a Plac familypromoter. In a particular embodiment, the lacO derivative is a lacOidsequence.

In a further embodiment, the present invention provides Pseudomonadorganisms that have been genetically modified to induce an auxotrophyand further modified to contain a chromosomal insertion of a nativeE.coli lacI gene, lacI^(Q) gene, or lacI^(Q1) gene other than as part ofa whole or truncated Plac-lacI-lacZYA operon. In another embodiment, thePseudomonad organism is further modified to contain a nucleic acidconstruct comprising at least one lacO sequence involved in therepression of transgene expression. In a particular embodiment, thePseudomonad organism is a Pseudomonas fluorescens.

In another aspect of the present invention, nucleic acid sequences areprovided for use in the improved production of proteins.

In one embodiment, nucleic acid sequences encoding prototrophy-restoringenzymes for use in an auxotrophic Pseudomonad host cells are provided.In a particular embodiment, nucleic acid sequences encoding nitrogenousbase compound biosynthesis enzymes purified from the organismPseudomonas fluorescens are provided. In one embodiment, nucleic acidsequences encoding the pyrF gene in Pseudomonas fluorescens is provided(SEQ. ID No.s 1 and 3). In another embodiment, a nucleic acid sequenceencoding the thyA gene in Pseudomonas fluorescens is provided (SEQ. ID.No. 4). In still another embodiment, nucleic acid sequences encoding anamino acid biosynthetic compound purified from the organism Pseudomonasfluorescens are provided. In a particular embodiment, a nucleic acidsequence encoding the proC gene in Pseudomonas fluorescens is provided(SEQ. ID No.s 6 and 8).

In another aspect, the present invention produces novel amino acidsequences which are the products of the novel nucleic acid expression.

In still another aspect of the present invention, nucleic acidconstructs are provided for use in the improved production of peptides.

In one embodiment, a nucleic acid construct for use in transforming aPseudomonad host cell comprising a) a nucleic acid sequence encoding arecombinant polypeptide, and b) a nucleic acid sequence encoding aprototrophy-enabling enzyme is provided. In another embodiment, thenucleic acid construct further comprises c) a Plac-Ptac family promoter.In still another embodiment, the nucleic acid construct furthercomprises d) at least one lacO sequence, or derivative, 3′ of a lac ortac family promoter. In yet another embodiment, the nucleic acidconstruct further comprises e) at least one lacO sequence, orderivative, 5′ of a lac or tac family promoter. In one embodiment, thederivative lacO sequence can be a lacOid sequence. In a particularembodiment, the Pseudomonad organism is Pseudomonas fluorescens.

In one embodiment of the present invention, nucleic acid constructs areprovided for use as expression vectors in Pseudomonad organismscomprising a) a nucleic acid sequence encoding a recombinantpolypeptide, b) a Plac-Ptac family promoter, c) at least one lacOsequence, or derivative, 3′ of a lac or tac family promoter, d) at leastone lacO sequence, or derivative, 5′ of a lac or tac family promoter. Inone embodiment, the derivative lacO sequence can be a lacOid sequence.In one embodiment, the nucleic acid construct further comprises e) aprototrophy-enabling selection marker for use in an auxotrophicPseudomonad cell. In a particular embodiment, the Pseudomonad organismis Pseudomonas fluorescens.

In another aspect of the present invention, modified cells are providedfor use in the improved production of proteins.

In one embodiment, an auxotrophic Pseudomonad cell is provided that hasa nucleic acid construct comprising i) a recombinant polypeptide, andii) a prototrophy-enabling nucleic acid. In another embodiment, thenucleic acid construct further comprises iii) a Plac-Ptac familypromoter. In still another embodiment, the nucleic acid constructfurther comprises iv) more than one lacO sequence. In one embodiment,the Pseudomonad is an auxotrophic Pseudomonas fluorescens cell. In afurther embodiment, the invention further comprises auxotrophicPseudomonad organisms, including Pseudomonas fluorescens, that have beenfurther genetically modified to contain a chromosomal insertion of anative E.coli lacI gene, lacI^(Q) gene, or lacI^(Q1) gene other than aspart of a whole or truncated Plac-lacI-lacZYA operon.

In another embodiment, a Pseudomonad cell is provided that comprises alacI transgene, or derivative thereof, other than as part of a whole ortruncated Plac-lacI-lacZYA operon, inserted into the chromosome, and b)a nucleic acid construct comprising i) a recombinant polypeptide, andii) a Plac-Ptac family promoter. In still another embodiment, thenucleic acid construct further comprises iii) at least one lacOsequence, and preferably, more than one lacO sequence. In oneembodiment, the lacO sequence is a lacOid sequence. In one embodiment,the Pseudomonad has been further modified to induce auxotrophy. In oneembodiment, the Pseudomonad cell is a Pseudomonas fluorescens.

In one aspect of the present invention, processes of expressingrecombinant polypeptides for use in improved protein production areprovided.

In one embodiment, the process provides expression of a nucleic acidconstruct comprising nucleic acids encoding a) a recombinantpolypeptide, and b) a prototrophy-restoring enzyme in a Pseudomonad thatis auxotrophic for at least one metabolite. In an alternativeembodiment, the Pseudomonad is auxotrophic for more than one metabolite.In one embodiment, the Pseudomonad is a Pseudomonas fluorescens cell. Ina particular embodiment, a recombinant polypeptide is expressed in aPseudomonad that is auxotrophic for a metabolite, or combination ofmetabolites, selected from the group consisting of a nitrogenous basecompound and an amino acid. In a more particular embodiment, recombinantpolypeptides are expressed in a Pseudomonad that is auxotrophic for ametabolite selected from the group consisting of uracil, proline, andthymidine. In another embodiment, the auxotrophy can be generated by theknock-out of the host pyrF, proC, or thyA gene, respectively. Analternative embodiment, recombinant polypeptides are expressed in anauxotrophic Pseudomonad cell that has been genetically modified throughthe insertion of a native E.coli lacI gene, lacI^(Q) gene, or lacI^(Q1)gene, other than as part of the PlacI-lacI-lacZYA operon, into the hostcell's chromosome. In one particular embodiment, the vector containingthe recombinant polypeptide expressed in the auxotroph comprises atleast one lacOid operator sequences. In one particular embodiment, thevector containing the recombinant polypeptide expressed in theauxotrophic host cell comprises at least two lac operator sequences, orderivatives thereof. In still a further embodiment, the recombinantpolypeptide is driven by a Plac family promoter.

In another embodiment, the process involves the use of Pseudomonad hostcells that have been genetically modified to provide at least one copyof a LacI encoding gene inserted into the Pseudomonad host cell'sgenome, wherein the lacI encoding gene is other than as part of thePlacI-lacI-lacZYA operon. In one embodiment, the gene encoding the Lacrepressor protein is identical to that of native E.coli lacI gene. Inanother embodiment, the gene encoding the Lac repressor protein is thelacI^(Q) gene. In still another embodiment, the gene encoding the Lacrepressor protein is the lacI^(Q1) gene. In a particular embodiment, thePseudomonad host cell is Pseudomonas fluorescens. In another embodiment,the Pseudomonad is further genetically modified to produce anauxotrophic cell. In another embodiment, the process producesrecombinant polypeptide levels of at least about 3 g/L, 4 g/L, 5 g/L 6g/L, 7 g/L, 8 g/L, 9 g/L or at least about 10 g/L. In anotherembodiment, the recombinant polypeptide is expressed in levels ofbetween 3 g/L and 100 g/L.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a comparison of the performance of P. fluorescensdual-plasmid expression systems using a pyrF marker (Δ and □) againstthe performance of P. fluorescens dual-plasmid expression systems usingonly antibiotic resistance markers (♦). All data shown are averages of9-multiple, representative 20-L fermentations, with IPTG being added toinduce target enzyme expression during mid-exponential phase. The upperset of three curves presents relative cell density data, which is readwith reference to the left vertical axis. The lower set of three curvespresents relative enzyme activity data for the target enzyme produced inthe corresponding fermentations, and is read with reference to the rightvertical axis. ♦—P. fluorescens containing pMYC plasmid having a tacpromoter-controlled target enzyme expression cassette and a tetracyclineresistance marker gene and containing a pCN plasmid having a lacIrepressor expression cassette and a kanamycin resistance marker gene.Variance bars shown are for these data points (n=4), and represent thenormal variance typically observed for this expression system amongdifferent fermentation runs. Δ—P. fluorescens strain with inactivatedgenomic pyrF containing pMYC plasmid having a tac promoter-controlledtarget enzyme expression cassette and a pyrF auxotrophic marker gene andcontaining pCN plasmid having a lacI repressor expression cassette and akanamycin resistance marker gene. □—P. fluorescens strain withinactivated genomic pyrF and proC containing pMYC plasmid having a tacpromoter-controlled target enzyme expression cassette and a pyrFauxotrophic marker gene and containing pCN plasmid having a lacIrepressor expression cassette and a proC auxotrophic marker gene.

FIG. 2 represents a map of the plasmid pDOW1249-2.

FIG. 3 represents a map of the plasmid pDOW1269-2.

FIG. 4 represents a schematic of lac operator constructs. LacZrepresents the positions of the native E.coli lacO sequences. tac DC239,DC240 represents the position of the native E.coli lac operator on aconstruct comprising a tac promoter and a nitrilase encoding nucleicacid. Opt lacO DC281 represents the position of the lacOid operatorsequence on a construct comprising a tac promoter and a nitrilaseencoding nucleic acid. Dual lacO DC262 represents the position of alacOid operator sequence 5′, and wild type lac operator sequence 3′ of atac promoter on a construct further comprising a nitrilase encodingnucleic acid.

FIG. 5 represents a Western Blot analysis (UnBlot) of LacI proteinaccumulation in the lacI integrant strains grown in a shake flask geneexpression medium. Broth samples were normalized to OD₆₀₀, combined withLDS NuPAGE sample buffer (Invitrogen), 50mM DTT and heated at 95° C. for40 min, then centrifuged briefly. Aliquots of 20 uL were loaded on a10%, 1 mm NuPAGE Bis-Tris gel run in MOPS with antioxidant in the innerchamber. Detection of the LacI protein was accomplished with an in-gelhybridization method (“UnBlot”, Pierce), using a polyclonal rabbitantibody to LacI (Stratagene cat. no. 217449-51) at 1:1000 and thesecondary antibody, Stabilized Goat Anti-rabbit Horseradish PeroxidaseConjugated Antibody (Pierce) at 1:500. The horseradish peroxidase wasvisualized with UnBlot Stable Peroxide and UnBlot Luminol Enhancer asaccording to the UnBlot kit.

FIG. 6 represents the composite of nitrilase accumulation profiles of DC140, DC239 and DC240. Data were compiled from DC140 (n=5), DC239 (n=5)and DC240 (n=4) runs. Dc140 is represented by ▪. DC239 is represented by□. DC240 is represented by □. Fermentation runs were performed over a 48hour period.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the Pseudomonad organisms have been geneticallymodified to induce an auxotrophy. In a particular embodiment, thePseudomonad organism is Pseudomonas fluorescens. In one embodiment, theauxotrophy is a result of genetic modifications to at least onenitrogenous base compound biosynthesis gene, or at least one amino acidbiosynthesis gene. In a further embodiment, the genetic modification isto a gene encoding an enzyme active in the uracil biosynthetic pathway,the thymidine biosynthetic pathway, or the proline biosynthetic pathway.In still a further embodiment, the genetic modification is to the pyrFgene encoding orotidine-5′-phosphate decarboxylase, the thyA geneencoding thymidilate synthase, or the proC gene encodingΔ¹-pyrroline-5-carboxylate reductase.

In another embodiment, the present invention provides Pseudomonadorganisms that have been genetically modified to provide at least onecopy of a LacI-encoding gene inserted into the genome, other than aspart of the PlacI-lacI-lacZYA operon. In a particular embodiment, thePseudomonad host cell is Pseudomonas fluorescens. In one embodiment, thePseudomonad contains a native E.coli lacI gene encoding the LacIrepressor protein.. In another embodiment, the Pseudomonad cell containsthe lacI^(Q) gene. In still another embodiment, the Pseudomonad cellcontains the lacI^(Q1) gene.

In another embodiment, a Pseudomonad organism is provided comprising anucleic acid construct containing a nucleic acid comprising at least onelacO sequence involved in the repression of transgene expression. In aparticular embodiment, the Pseudomonad host cell is Pseudomonadfluorescens. In one embodiment, the nucleic acid construct comprisesmore than one lacO sequence. In another embodiment, the nucleic acidconstruct comprises at least one, and preferably more than one, lacOidsequence. In one embodiment, the nucleic acid construct comprises a lacOsequence, or derivative thereof, located 3′ of a Plac family promoter,and a lacO sequence, or derivative thereof, located 5′ of a Plac familypromoter. In a particular embodiment, the lacO derivative is a lacOidsequence.

In a further embodiment, the present invention provides Pseudomonadorganisms that have been genetically modified to induce an auxotrophyand further modified to contain a chromosomal insertion of a nativeE.coli lacI gene, lacI Q gene, or lacIQ1 gene other than as part of awhole or truncated Plac-lacI-lacZYA operon. In another embodiment, thePseudomonad organism is further modified to contain a nucleic acidconstruct comprising at least one lacO sequence involved in therepression of transgene expression. In a particular embodiment, thePseudomonad organism is a Pseudomonas fluorescens.

The host cell provided by the present invention for use in an expressionsystem producing recombinant polypeptides can be selected from the“Pseudomonads and closely related bacteria” or from a Subgroup thereof,as defined below. In one embodiment, the host cell is selected from thegenus Pseudomonas. In a particular embodiment, the particular species ofPseudomonas is P. fluorescens. In a particular embodiment, the host cellis Pseudomonas fluorescens biotype A or biovar I.

Definitions

The term “isolated” refers to nucleic acid, protein, or peptide that issubstantially or essentially free from other material components, forexample, which can be cellular components.

The term “fragment” means a portion or partial sequence of a nucleotide,protein, or peptide sequence.

As used herein, the term “percent total cell protein” means the amountof protein or peptide in the host cell as a percentage of aggregatecellular protein.

The term “operably attached,” as used herein, refers to anyconfiguration in which the transcriptional and any translationalregulatory elements are covalently attached to the encoding sequence insuch disposition(s), relative to the coding sequence, that in and byaction of the host cell, the regulatory elements can direct theexpression of the coding sequence.

The term “auxotrophic,” as used herein, refers to a cell which has beenmodified to eliminate or reduce its ability to produce a specificsubstance required for growth and metabolism.

As used herein, the term “percent total cell protein” means a measure ofthe fraction of total cell protein that represents the relative amountof a given protein expressed by the cell.

The term “prototrophy,” as used herein, refers to a cell that is capableof producing a specific substance required for growth and metabolism.

As used herein, the term “homologous” or means either i) a protein orpeptide that has an amino acid sequence that is substantially similar(i.e., at least 70, 75, 80, 85, 90, 95, or 98%) to the sequence of agiven original protein or peptide and that retains a desired function ofthe original protein or peptide or ii) a nucleic acid that has asequence that is substantially similar (i.e., at least 70, 75, 80, 5,90, 95, or 98%) to the sequence of a given nucleic acid and that retainsa desired function of the original nucleic acid sequence. In all of theembodiments of this invention and disclosure, any disclosed protein,peptide or nucleic acid can be substituted with a homologous orsubstantially homologous protein, peptide or nucleic acid that retains adesired function. In all of the embodiments of this invention anddisclosure, when any nucleic acid is disclosed, it should be assumedthat the invention also includes all nucleic acids that hybridize to thedisclosed nucleic acid.

In one non-limiting embodiment, the non-identical amino acid sequence ofthe homologous polypeptide can be amino acids that are members of anyone of the 15 conservative or semi-conservative groups shown in Table 1.TABLE 1 SIMILAR AMINO ACID SUBSTITUTION GROUPS Semi-ConservativeConservative Groups (8) Groups (7) Arg, Lys Arg, Lys, His Asp, Glu Asn,Asp, Glu, Gln Asn, Gln Ile, Leu, Val Ile, Leu, Val, Met, Phe Ala, GlyAla, Gly, Pro, Ser, Thr Ser, Thr Ser, Thr, Tyr Phe, Tyr Phe, Trp, TyrCys (non-cystine), Ser Cys (non-Cystine), Ser, Thr

Amino acid sequences provided herein are represented by the followingabbreviations: A Ala alanine P Pro proline B aspartate or asparagine QGln glutamine C Cys cysteine R Arg arginine D Asp aspartate S Ser serineE Glu glutamate T Thr threonine F Phe phenylalanine G Gly glycine V Valvaline H His histidine W Trp tryptophan I Ile isoleucine Y Tyr tyrosineZ glutamate or glutamine K Lys lysine L Leu leucine M Met methionine NAsn asparagine

I. Selection of Pseudomonads and Related Bacteria as Host Cells

The present invention provides the use of Pseudomonads and relatedbacteria as host cells in the improved production of proteins.

Auxotrophic Selection Efficiency

It has been discovered that Pseudomonads have the ability to utilizeauxotrophic selection markers for the maintenance of protein expressingplasmids without the drawbacks typically associated with other systems,such as plasmid instability and cross-feeding.

Auxotrophic markers, in other host cell systems, are not alwayssufficient to maintain plasmids in every cell, especially duringfermentations where loss of the plasmid may give plasmid-less cells aselective advantage, resulting in the accumulation of a large fractionof nonproductive cells, reducing product formation. Such revertantstrains are especially troublesome if they have the ability tocross-feed the auxotrophic metabolite from prototrophic enabledbacteria. For example, use of the trp operon on a plasmid in an E.colitryptophan auxotroph was not sufficient to prevent a large proportion ofplasmid-less cells from accumulating, until combined with the valS gene(encoding valyl t-RNA synthetase) in a valS^(ts) host ( Skogman, S. G.;Nilsson, J., Temperature-dependent retention of atryptophan-operon-bearing plasmid in Escherichia coli. Gene 1984, 31,(1-3), 117-22.) Presumably, the cells containing the trp operon on aplasmid secreted enough tryptophan or related molecules to allow growthof plasmid-less cells. Likewise, using the LEU2 gene on axylitol-reductase production plasmid in leu2 mutant yeast resulted inplasmid loss; up to 80% of a fed-batch culture was made up of cellswithout a production plasmid, because leucine was secreted byplasmid-containing cells into the broth (Meinander, N. Q.;Hahn-Haegerdal, B., Fed-batch xylitol production with two recombinantSaccharomyces cerevisiae strains expressing XYL1 at different levels,using glucose as a cosubstrate: a comparison of production parametersand strain stability. Biotechnology and Bioengineering 1997, 54, (4),391-399).

It has been discovered that Pseudomonas fluorescens (Pf) does notexhibit the inherent problems associated with cross-feeding observed inother host cell systems, for example, E.coli and yeast. While notwanting to be bound by any particular theory, it is thought thatauxotrophic Pseudomonas fluorescens is a particularly suitable organismfor use as a host cell because of the observed inability of a Pfauxotrophic cell to out compete a auxotrophic cell containing aprototrophic-enabling plasmid on a supplemented medium that contains theauxotrophic metabolite, indicating an innate difficulty of an Pfauxotroph to import the required metabolite. Because of this, Pfauxotrophic cells that lose the selection marker plasmid do not gain aselective advantage over Pf auxotrophic cells containing the selectionmarker, even in the presence of a supplemental metabolite, greatlyreducing any potential effects of cross-feeding. Because of the reducedeffects of cross-feeding, production yields of the recombinantpolypeptide in a fermentation run are not reduced due to the presence ofnon-recombinant polypeptide producing cells.

LacI Insert

It has been discovered that Pseudomonads are able to use a single-copylacI transgene, other than as part of a whole or truncatedPlac-lacI-lacZYA operon, chromosomal insert to effectively repressprotein expression until induction.

Transcription initiation from regulated promoters by RNA polymerase isactivated or deactivated by the binding or releasing of a regulatoryprotein. Thus, regulated promoters include those that participate innegative control (i.e. repressible promoters), wherein the gene encodingthe target polypeptide of interest is expressed only when the promoteris free of the regulator protein (i.e. a “repressor” protein), and thosethat participate in positive control, wherein the gene is expressed onlywhen the promoter is bound by the regulator protein (i.e. an “activator”protein).

One of the most common classes of repressible promoters used inbacterial expression systems is the family of Plac-based promoters. Thefamily of Plac-based promoters originates with the native E.coli lactoseoperon, referred to as the “lac” operon, also symbolized as “lacZYA,”the expression of which is regulated by the expression product of thelacI gene. The native E.coli structure of the operon is“PlacI-lacI-PlacZ-lacZYA,” wherein the native E.coli Plac promoter isrepresented by “PlacZ” (also called “PlacZYA”). “PlacI” represents thenative promoter for the lacI gene, and “lacI” represents the geneencoding the lac repressor, i.e. the LacI protein. “lacZYA” representsthe operon encoding the lactose utilization pathway.

The LacI-regulated promoters include, among others, the native E.colilactose operon promoter (“Plac”). In addition, improved mutants havealso been discovered, as have intra promoter hybrids of Plac, such asthe “Ptac” promoter, “Ptrc” promoter, and the “PtacII” promoters. ThePtac promoter in E.coli, for example, is 3-fold stronger than the Placpromoter when fully derepressed. Therefore, it is frequently used forpromoting high level regulated gene expression in E.coli. However, whilethe Plac promoter is 1,000-fold repressed by LacI, while the Ptacpromoter is only 50-fold repressed under similar conditions (Lanzer, M.& H. Bujard. 1988. Proc. Natl. Acad. Sci. USA. 85:8973). Repression ofthe E. coli Ptac promoter or other lac related promoters, depends uponthe concentration of the repressor, LacI. (De Boer, et al., 1983. Proc.Natl. Acad. Sci. USA. 78:21-25). As set forth above, release fromrepression can occur through the addition of an inducer which reducesthe affinity of the repressor for its specific DNA binding site, in thiscase, the lac operator (lacO). Alternatively, a reduction in theconcentration of the repressor relative to the molar concentration ofspecific DNA binding sites on the plasmid can also derepress thepromoter. If the lacI gene is located on a high copy number cloningplasmid, then a large amount of inducer is required to initiateexpression because of the large amount of repressor produced in such asystem.

In commercial production systems, the lac repressor is typically encodedby a gene whose expression is constitutive, i.e. non-regulated, thusproviding an intracellular environment in which the desired transgene,encoding the desired target protein, is repressed until a desired hostcell biomass or cell density is achieved. At that time, a quantity of asmall molecule known as an inducer whose presence is effective todissociate the repressor from the transgene, is added to the cellculture and taken up by the host cell, thereby permitting transcriptionof the transgene. In the case of lac repressor proteins, the inducer canbe lactose or a non-metabolized, gratuitous inducer such asisopropyl-beta-D-thio-galactoside (“IPTG”). The selected point in timeat which the inducer is to be added is referred to as the “inductionphase.”

A variety of lac repressor genes have been identified as useful for therepression of Plac family promoters present on recombinant polypeptideexpression vectors. These include the native E.coli lacI gene and/or byvariants thereof, including the lacI^(Q) and lacI^(Q1) genes that encodethe same LacI protein, but at a higher expression level. For example,the lacI^(Q) mutation is a single CG to TA change at -35 of the promoterregion of lacI (Calos, M. 1978. Nature 274:762) which causes a 10-foldincrease in LacI expression in E.coli (Mueller-Hill, B., et al. 1968.Proc. Natl. Acad. Sci. USA. 59:1259). Wild-type E.coli cells have aconcentration of LacI of 10⁻⁸ M or about 10 molecules per cell, with 99%of the protein present as a tetramer (Fickert, R. & B. Mueller-Hill1992. J. Mol. Biol. 226:59). Cells containing the lacI^(Q) mutationcontain about 100 molecules per cell or 10⁻⁷ M LacI. As a result, anumber of bacterial expression systems have been developed in which Placfamily promoter controlled transgenes, resident in plasmids, aremaintained in host cells expressing LacI proteins at different levels,thereby repressing the desired transgene until a chosen “inductionphase” of cell growth.

In many cases, however, repression of expression of the target proteinduring cell growth can be imperfect, resulting in a significant amountof expression prior to the particular induction phase. This “leaky”repression results in host cell stress, inefficient utilization ofcarbon source due to metabolic energy being diverted from normal cellgrowth to transgene, and a delay in reaching optimal cell densityinduction points, resulting in a more lengthy and costly fermentationrun, and often, a reduced yield of the target protein.

One common strategy for improving repression of Plac-familypromoter-driven transgenes has been to place a lacI or a lacI^(Q) geneon the plasmid bearing the Plac-family promoter-driven target gene (e.g.see MJR Stark in Gene 51:255-67 (1987) and E Amann et al. in Gene:301-15(1988)). However, this often results in overproduction of the Lacrepressor protein, which then requires use of an even higher inducerconcentration to restore induction levels of the transgene to overcomethe decrease in recombinant protein production. Moreover, the use of asecond plasmid containing the lacI gene, separate from the plasmidcontaining the Plac-family promoter-driven target gene, requires the useof two different selection marker genes in order to maintain bothplasmids in the expression host cell: one selection marker gene for eachof the two different plasmids. The presence of the second selectionmarker gene, i.e. the selection marker gene for the second plasmid, inturn requires the use of either: 1) a separate antibiotic in the case ofan antibiotic-resistance selection marker gene, which is costly anddisadvantageous from a health/safety regulatory perspective; or 2) aseparate metabolic deficiency in the host cell genome, in the case of anauxotrophic selection marker gene, which requires the additional work ofmutating the host cell.

It has surprisingly been discovered that a lacI insertion, other than aspart of a whole or truncated Plac-lacI-lacZYA operon, is as effective inrepressing Plac-Ptac family promoter-controlled transgenes as amulti-copy plasmid encoding a LacI repressor protein in Pseudomonasfluorescens. This surprising discovery eliminates the need to maintain aseparate plasmid encoding a LacI repressor protein in the cell, oreliminates the need to define an additional auxotrophic selectionmarker, and further reduces the potential production inefficienciescaused by such maintenance of a lacI containing plasmid.

In a previous attempt to regulate transgene expression in Pseudomonas,an E.coli PlacI-lacI-lacZYA operon that has been deleted of the lacZpromoter region, but that retains the constitutive PlacI promoter, waschromosomally inserted (See U.S. Pat. No. 5,169,760). The deletionallows for constitutive expression of the gene products of the lacoperon. However, the inserted operon contains the E.coli lacy gene,which encodes for the lactose transporter protein lactose permease.Lactose permease is capable of transporting lactose, or similarderivatives, into the host cell from the medium. The presence of lactosepermease may lead to increased importation of lactose-like contaminantsfrom the medium, ultimately resulting in derepression of the Plac familypromoter prior to induction. Furthermore, expression of the lac operonlacZ, lacY, and lacA gene products may result in the inefficientdedication of carbon utilization resources to these products, resultingin increased metabolic stress on the cells, and delaying theestablishment of a high cell density for induction. In addition, thelarger lacI-lacZYA fusion operon may produce increased messageinstability compared to a lacI insert alone in a host cell.

It has been surprisingly discovered that the use of a LacI-encoding geneother than as part of a whole or truncated PlacI-lacI-lacZYA operon inPseudomonads surprisingly resulted in substantially improved repressionof pre-induction recombinant protein expression, higher cell densitiesin commercial-scale fermentation, and higher yields of the desiredproduct in comparison with previously taught lacI-lacZYA Pseudomonadchromosomal insertion (U.S. Pat. No. 5,169,760).

Additional attempts to utilize derivative lacI genes, such as lacI^(Q)and lacI^(Q1,) which are expressed at greater levels than lacI due topromoter modifications, have also been described. C G Glascock & M JWeickert describe E.coli strains in which a separate LacIprotein-encoding gene was present in the chromosome of the host cell inan attempt to assess the level of control of a plasmid-borne Ptac-driventarget gene. See C G Glascock & M J Weickert, “Using chromosomallacI^(Q1) to control expression of genes on high-copy number plasmids inEscherichia coli,” Gene 223(1-2):221-31(1998); See also WO 97/04110.Among the LacI protein-encoding genes tested were lacI, lacI^(Q), andlacI^(Q1). The results obtained for the lacI gene and the lacI^(Q) genedemonstrated inferior levels of repression of the Ptac-driven targetgene when present on a high-copy number plasmid, resulting insubstantial levels of pre-induction target gene expression. Only thehigh expressing lacI^(Q1) gene provided sufficient repression in thatsystem.

Such a strategy, however, has the potential to increase costs byincreasing the amount of inducer required to sufficiently derepress thepromoter at induction, and decreasing yields due to the inability of theinducer to sufficiently bind all of the constitutively expressedrepressor protein.

Comparatively, it has surprisingly been discovered that a single-copylacI chromosomal insert was sufficient to repress Plac-Ptac familypromoter driven transgene expression. Such a discovery allows potentialcost saving measures on the amount of inducer used, and providesadditional flexibility in the development of Pseudomonas fluorescens asa host cell in the improved production of proteins.

Pseudomonas Organisms

Pseudomonads and closely related bacteria, as used herein, isco-extensive with the group defined herein as “Gram(−) ProteobacteriaSubgroup 1.”“Gram(−) Proteobacteria Subgroup 1” is more specificallydefined as the group of Proteobacteria belonging to the families and/orgenera described as falling within that taxonomic “Part” named“Gram-Negative Aerobic Rods and Cocci” by R. E. Buchanan and N. E.Gibbons (eds.), Bergey's Manual of Determinative Bacteriology, pp.217-289 (8th ed., 1974) (The Williams & Wilkins Co., Baltimore, Md.,USA) (hereinafter “Bergey (1974)”). Table 4 presents the families andgenera of organisms listed in this taxonomic “Part.” TABLE 1 FAMILIESAND GENERA LISTED IN THE PART, “GRAM- NEGATIVE AEROBIC RODS AND COCCI”(IN BERGEY (1974)) Family I. Pseudomonadaceae Gluconobacter PseudomonasXanthomonas Zoogloea Family II. Azotobacteraceae Azomonas AzotobacterBeijerinckia Derxia Family III. Rhizobiaceae Agrobacterium RhizobiumFamily IV. Methylomonadaceae Methylococcus Methylomonas Family V.Halobacteriaceae Halobacterium Halococcus Other Genera AcetobacterAlcaligenes Bordetella Brucella Francisella Thermus

“Gram(−) Proteobacteria Subgroup 1”contains all Proteobacteriaclassified there under, as well as all Proteobacteria that would beclassified according to the criteria used in forming that taxonomic“Part.” As a result, “Gram(−) Proteobacteria Subgroup 1” excludes, e.g.:all Gram-positive bacteria; those Gram-negative bacteria, such as theEnterobacteriaceae, which fall under others of the 19 “Parts” of thisBergey (1974) taxonomy; the entire “Family V. Halobacteriaceae” of thisBergey (1974) “Part,” which family has since been recognized as being anon-bacterial family of Archaea; and the genus, Thermus, listed withinthis Bergey (1974) “Part,” which genus which has since been recognizedas being a non-Proteobacterial genus of bacteria. “Gram(−)Proteobacteria Subgroup 1” further includes those Proteobacteriabelonging to (and previously called species of) the genera and familiesdefined in this Bergey (1974) “Part,” and which have since been givenother Proteobacterial taxonomic names. In some cases, these re-namingsresulted in the creation of entirely new Proteobacterial genera. Forexample, the genera Acidovorax, Brevundimonas, Burkholderia,Hydrogenophaga, Oceanimonas, Ralstonia, and Stenotrophomonas, werecreated by regrouping organisms belonging to (and previously calledspecies of) the genus Pseudomonas as defined in Bergey (1974). Likewise,e.g., the genus Sphingomonas (and the genus Blastomonas, derivedtherefrom) was created by regrouping organisms belonging to (andpreviously called species of) the genus Xanthomonas as defined in Bergey(1974). Similarly, e.g., the genus Acidomonas was created by regroupingorganisms belonging to (and previously called species of) the genusAcetobacter as defined in Bergey (1974). Such subsequently reassignedspecies are also included within “Gram(−) Proteobacteria Subgroup 1” asdefined herein.

In other cases, Proteobacterial species falling within the genera andfamilies defined in this Bergey (1974) “Part” were simply reclassifiedunder other, existing genera of Proteobacteria. For example, in the caseof the genus Pseudomonas, Pseudomonas enalia (ATCC 14393), Pseudomonasnigrifaciens (ATCC 19375), and Pseudomonas putrefaciens (ATCC 8071) havesince been reclassified respectively as Alteromonas haloplanktis,Alteromonas nigrifaciens, and Alteromonas putrefaciens. Similarly, e.g.,Pseudomonas acidovorans (ATCC 15668) and Pseudomonas testosteroni (ATCC11996) have since been reclassified as Comamonas acidovorans andComamonas testosteroni, respectively; and Pseudomonas nigrifaciens (ATCC19375) and Pseudomonas piscicida (ATCC 15057) have since beenreclassified respectively as Pseudoalteromonas nigrifaciens andPseudoalteromonas piscicida. Such subsequently reassignedProteobacterial species are also included within “Gram(−) ProteobacteriaSubgroup 1” as defined herein. “Gram(−) Proteobacteria Subgroup 1” alsoincludes Proteobacterial species that have since been discovered, orthat have since been reclassified as belonging, within theProteobacterial families and/or genera of this Bergey (1974) “Part.” Inregard to Proteobacterial families, “Gram(−) Proteobacteria Subgroup 1”also includes Proteobacteria classified as belonging to any of thefamilies: Pseudomonadaceae, Azotobacteraceae (now often called by thesynonym, the “Azotobacter group” of Pseudomonadaceae), Rhizobiaceae, andMethylomonadaceae (now often called by the synonym, “Methylococcaceae”).Consequently, in addition to those genera otherwise described herein,further Proteobacterial genera falling within “Gram(−) ProteobacteriaSubgroup 1” include: 1) Azotobacter group bacteria of the genusAzorhizophilus; 2) Pseudomonadaceae family bacteria of the generaCellvibrio, Oligella, and Teredinibacter; 3) Rhizobiaceae familybacteria of the genera Chelatobacter, Ensifer, Liberibacter (also called“Candidatus Liberibacter”), and Sinorhizobium; and 4) Methylococcaceaefamily bacteria of the genera Methylobacter, Methylocaldum,Methylomicrobium, Methylosarcina, and Methylosphaera.

In one embodiment, the host cell is selected from “Gram(−)Proteobacteria Subgroup 1,”as defined above.

In another embodiment, the host cell is selected from “Gram(−)Proteobacteria Subgroup 2.” “Gram(−) Proteobacteria Subgroup 2” isdefined as the group of Proteobacteria of the following genera (with thetotal numbers of catalog-listed, publicly-available, deposited strainsthereof indicated in parenthesis, all deposited at ATCC, except asotherwise indicated): Acidomonas (2); Acetobacter (93); Gluconobacter(37); Brevundimonas (23); Beijerinckia (13); Derxia (2); Brucella (4);Agrobacterium (79); Chelatobacter (2); Ensifer (3); Rhizobium (144);Sinorhizobium (24); Blastomonas (1); Sphingomonas (27); Alcaligenes(88); Bordetella (43); Burkholderia (73); Ralstonia (33); Acidovorax(20); Hydrogenophaga (9); Zoogloea (9); Methylobacter (2); Methylocaldum(1 at NCIMB); Methylococcus (2); Methylomicrobium (2); Methylomonas (9);Methylosarcina (1); Methylosphaera; Azomonas (9); Azorhizophilus (5);Azotobacter (64); Cellvibrio (3); Oligella (5); Pseudomonas (1139);Francisella (4); Xanthomonas (229); Stenotrophomonas (50); andOceanimonas (4).

Exemplary host cell species of “Gram(−) Proteobacteria Subgroup 2”include, but are not limited to the following bacteria (with the ATCC orother deposit numbers of exemplary strain(s) thereof shown inparenthesis): Acidomonas methanolica (ATCC 43581); Acetobacter aceti(ATCC 15973); Gluconobacter oxydans (ATCC 19357); Brevundimonas diminuta(ATCC 11568); Bejerinckia indica (ATCC 9039 and ATCC 19361); Derxiagummosa (ATCC 15994); Brucella melitensis (ATCC 23456), Brucella abortus(ATCC 23448); Agrobacterium tumefaciens (ATCC 23308), Agrobacteriumradiobacter (ATCC 19358), Agrobacterium rhizogenes (ATCC 11325);Chelatobacter heintzii (ATCC 29600); Ensifer adhaerens (ATCC 33212);Rhizobium leguminosarum (ATCC 10004); Sinorhizobium fredii (ATCC 35423);Blastomonas natatoria (ATCC 35951); Sphingomonas paucimobilis (ATCC29837); Alcaligenes faecalis (ATCC 8750); Bordetella pertussis (ATCC9797); Burkholderia cepacia (ATCC 25416); Ralstonia pickettii (ATCC27511); Acidovoraxfacilis (ATCC 11228); Hydrogenophaga flava (ATCC33667); Zoogloea ramigera (ATCC 19544); Methylobacter luteus (ATCC49878); Methylocaldum gracile (NCIMB 11912); Methylococcus capsulatus(ATCC 19069); Methylomicrobium agile (ATCC 35068); Methylomonasmethanica (ATCC 35067); Methylosarcina fibrata (ATCC 700909);Methylosphaera hansonii (ACAM 549); Azomonas agilis (ATCC 7494);Azorhizophilus paspali (ATCC 23833); Azotobacter chroococcum (ATCC9043); Cellvibrio mixtus (UQM 2601); Oligella urethralis (ATCC 17960);Pseudomonas aeruginosa (ATCC 10145), Pseudomonas fluorescens (ATCC35858); Francisella tularensis (ATCC 6223); Stenotrophomonas maltophilia(ATCC 13637); Xanthomonas campestris (ATCC 33913); and Oceanimonasdoudoroffii (ATCC 27123).

In another embodiment, the host cell is selected from “Gram(−)Proteobacteria Subgroup 3.” “Gram(−) Proteobacteria Subgroup 3” isdefined as the group of Proteobacteria of the following genera:Brevundimonas; Agrobacterium; Rhizobium; Sinorhizobium; Blastomonas;Sphingomonas ; Alcaligenes; Burkholderia; Ralstonia; Acidovorax;Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus;Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera;Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella;Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas;and Oceanimonas.

In another embodiment, the host cell is selected from “Gram(−)Proteobacteria Subgroup 4.” “Gram(−) Proteobacteria Subgroup 4” isdefined as the group of Proteobacteria of the following genera:Brevundimonas; Blastomonas; Sphingomonas; Burkholderia; Ralstonia;Acidovorax; Hydrogenophaga; Methylobacter; Methylocaldum; Methylococcus;Methylomicrobium; Methylomonas; Methylosarcina; Methylosphaera;Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella;Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas; Xanthomonas;and Oceanimonas.

In an embodiment, the host cell is selected from “Gram(−) ProteobacteriaSubgroup 5.” “Gram(−) Proteobacteria Subgroup 5” is defined as the groupof Proteobacteria of the following genera: Methylobacter; Methylocaldum;Methylococcus; Methylomicrobium; Methylomonas; Methylosarcina;Methylosphaera; Azomonas; Azorhizophilus; Azotobacter; Cellvibrio;Oligella; Pseudomonas; Teredinibacter; Francisella; Stenotrophomonas;Xanthomonas; and Oceanimonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 6.”“Gram(−) Proteobacteria Subgroup 6” is defined as the group ofProteobacteria of the following genera: Brevundimonas; Blastomonas;Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga;Azomonas; Azorhizophilus; Azotobacter; Cellvibrio; Oligella;Pseudomonas; Teredinibacter; Stenotrophomonas; Xanthomonas; andOceanimonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 7.”“Gram(−) Proteobacteria Subgroup 7” is defined as the group ofProteobacteria of the following genera: Azomonas; Azorhizophilus;Azotobacter; Cellvibrio; Oligella; Pseudomonas; Teredinibacter;Stenotrophomonas; Xanthomonas; and Oceanimonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 8.”“Gram(−) Proteobacteria Subgroup 8” is defined as the group ofProteobacteria of the following genera: Brevundimonas; Blastomonas;Sphingomonas; Burkholderia; Ralstonia; Acidovorax; Hydrogenophaga;Pseudomonas; Stenotrophomonas; Xanthomonas; and Oceanimonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 9.”“Gram(−) Proteobacteria Subgroup 9” is defined as the group ofProteobacteria of the following genera: Brevundimonas; Burkholderia;Ralstonia; Acidovorax; Hydrogenophaga; Pseudomonas; Stenotrophomonas;and Oceanimonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 10.”“Gram(−) Proteobacteria Subgroup 10” is defined as the group ofProteobacteria of the following genera: Burkholderia; Ralstonia;Pseudomonas; Stenotrophomonas; and Xanthomonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 11.”“Gram(−) Proteobacteria Subgroup 11” is defined as the group ofProteobacteria of the genera: Pseudomonas; Stenotrophomonas; andXanthomonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 12.”“Gram(−) Proteobacteria Subgroup 12” is defined as the group ofProteobacteria of the following genera: Burkholderia; Ralstonia;Pseudomonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 13.”“Gram(−) Proteobacteria Subgroup 13” is defined as the group ofProteobacteria of the following genera: Burkholderia; Ralstonia;Pseudomonas; and Xanthomonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 14.”“Gram(−) Proteobacteria Subgroup 14” is defined as the group ofProteobacteria of the following genera: Pseudomonas and Xanthomonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 15.”“Gram(−) Proteobacteria Subgroup 15” is defined as the group ofProteobacteria of the genus Pseudomonas.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 16.” “Gram(−) Proteobacteria Subgroup 16” is defined as the group ofProteobacteria of the following Pseudomonas species (with the ATCC orother deposit numbers of exemplary strain(s) shown in parenthesis):Pseudomonas abietaniphila (ATCC 700689); Pseudomonas aeruginosa (ATCC10145); Pseudomonas alcaligenes (ATCC 14909); Pseudomonasanguilliseptica (ATCC 33660); Pseudomonas citronellolis (ATCC 13674);Pseudomonasflavescens (ATCC 51555); Pseudomonas mendocina (ATCC 25411);Pseudomonas nitroreducens (ATCC 33634); Pseudomonas oleovorans (ATCC8062); Pseudomonas pseudoalcaligenes (ATCC 17440); Pseudomonasresinovorans (ATCC 14235); Pseudomonas straminea (ATCC 33636);Pseudomonas agarici (ATCC 25941); Pseudomonas alcaliphila; Pseudomonasalginovora; Pseudomonas andersonii; Pseudomonas asplenii (ATCC 23835);Pseudomonas azelaica (ATCC 27162); Pseudomonas beijerinckii (ATCC19372); Pseudomonas borealis; Pseudomonas boreopolis (ATCC 33662);Pseudomonas brassicacearum; Pseudomonas butanovora (ATCC 43655);Pseudomonas cellulosa (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663);Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, ATCC 17418, ATCC17461); Pseudomonas fragi (ATCC 4973); Pseudomonas lundensis (ATCC49968); Pseudomonas taetrolens (ATCC 4683); Pseudomonas cissicola (ATCC33616); Pseudomonas coronafaciens; Pseudomonas diterpeniphila;Pseudomonas elongata (ATCC 10144); Pseudomonas flectens (ATCC 12775);Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella;Pseudomonas corrugata (ATCC 29736); Pseudomonas extremorientalis;Pseudomonas fluorescens (ATCC 35858); Pseudomonas gessardii; Pseudomonaslibanensis; Pseudomonas mandelii (ATCC 700871); Pseudomonas marginalis(ATCC 10844); Pseudomonas migulae; Pseudomonas mucidolens (ATCC 4685);Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha(ATCC 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas veronii(ATCC 700474); Pseudomonas frederiksbergensis; Pseudomonas geniculata(ATCC 19374); Pseudomonas gingeri; Pseudomonas graminis; Pseudomonasgrimontii; Pseudomonas halodenitrificans; Pseudomonas halophila;Pseudomonas hibiscicola (ATCC 19867); Pseudomonas huttiensis (ATCC14670); Pseudomonas hydrogenovora; Pseudomonas jessenii (ATCC 700870);Pseudomonas kilonensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonaslini; Pseudomonas marginata (ATCC 25417); Pseudomonas mephitica (ATCC33665); Pseudomonas denitrificans (ATCC 19244); Pseudomonaspertucinogena (ATCC 190); Pseudomonas pictorum (ATCC 23328); Pseudomonaspsychrophila; Pseudomonas fulva (ATCC 31418); Pseudomonas monteilii(ATCC 700476); Pseudomonas mosselii; Pseudomonas oryzihabitans (ATCC43272); Pseudomonas plecoglossicida (ATCC 700383); Pseudomonas putida(ATCC 12633); Pseudomonas reactans; Pseudomonas spinosa (ATCC 14606);Pseudomonas balearica; Pseudomonas luteola (ATCC 43273); Pseudomonasstutzeri (ATCC 17588); Pseudomonas amygdali (ATCC 33614); Pseudomonasavellanae (ATCC 700331); Pseudomonas caricapapayae (ATCC 33615);Pseudomonas cichorii (ATCC 10857); Pseudomonas fcuserectae (ATCC 35104);Pseudomonas fuscovaginae; Pseudomonas meliae (ATCC 33050); Pseudomonassyringae (ATCC 19310); Pseudomonas viridiflava (ATCC 13223); Pseudomonasthermocarboxydovorans (ATCC 35961); Pseudomonas thermotolerans;Pseudomonas thivervalensis; Pseudomonas vancouverensis (ATCC 700688);Pseudomonas wisconsinensis; and Pseudomonas xiamenensis.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 17.”“Gram(−) Proteobacteria Subgroup 17” is defined as the group ofProteobacteria known in the art as the “fluorescent Pseudomonads”including those belonging, e.g., to the following Pseudomonas species:Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella;Pseudomonas corrugata; Pseudomonas extremorientalis; Pseudomonasfluorescens; Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonasmandelii; Pseudomonas marginalis; Pseudomonas migulae; Pseudomonasmucidolens; Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonassynxantha; Pseudomonas tolaasii; and Pseudomonas veronii.

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 18.”“Gram(−) Proteobacteria Subgroup 18” is defined as the group of allsubspecies, varieties, strains, and other sub-special units of thespecies Pseudomonas fluorescens, including those belonging, e.g., to thefollowing (with the ATCC or other deposit numbers of exemplary strain(s)shown in parenthesis): Pseudomonas fluorescens biotype A, also calledbiovar 1 or biovar I (ATCC 13525); Pseudomonas fluorescens biotype B,also called biovar 2 or biovar II (ATCC 17816); Pseudomonas fluorescensbiotype C, also called biovar 3 or biovar III (ATCC 17400); Pseudomonasfluorescens biotype F, also called biovar 4 or biovar IV (ATCC 12983);Pseudomonas fluorescens biotype G, also called biovar 5 or biovar V(ATCC 17518); Pseudomonas fluorescens biovar VI; Pseudomonas fluorescensPf0-1; Pseudomonas fluorescens Pf-5 (ATCC BAA-477); Pseudomonasfluorescens SBW25; and Pseudomonas fluorescens subsp. cellulosa (NCIMB10462).

The host cell can be selected from “Gram(−) Proteobacteria Subgroup 19.”“Gram(−) Proteobacteria Subgroup 19” is defined as the group of allstrains of Pseudomonas fluorescens biotype A. A particularly particularstrain of this biotype is P. fluorescens strain MB101 (see U.S. Pat. No.5,169,760 to Wilcox), and derivatives thereof.

In one embodiment, the host cell is any of the Proteobacteria of theorder Pseudomonadales. In a particular embodiment, the host cell is anyof the Proteobacteria of the family Pseudomonadaceae.

In a particular embodiment, the host cell is selected from “Gram(−)Proteobacteria Subgroup 1. ” In a particular embodiment, the host cellis selected from “Gram(−) Proteobacteria Subgroup 2. ” In a particularembodiment, the host cell is selected from “Gram(−) ProteobacteriaSubgroup 3. ” In a particular embodiment, the host cell is selected from“Gram(−) Proteobacteria Subgroup 5. ” In a particular embodiment, thehost cell is selected from “Gram(−) Proteobacteria Subgroup 7. ” In aparticular embodiment, the host cell is selected from “Gram(−)Proteobacteria Subgroup 12. ” In a particular embodiment, the host cellis selected from “Gram(−) Proteobacteria Subgroup 15. ” In a particularembodiment, the host cell is selected from “Gram(−) ProteobacteriaSubgroup 17. ” In a particular embodiment, the host cell is selectedfrom “Gram(−) Proteobacteria Subgroup 18. ” In a particular embodiment,the host cell is selected from “Gram(−) Proteobacteria Subgroup 19. ”

Additional P. fluorescens strains that can be used in the presentinvention include Pseudomonas fluorescens Migula and Pseudomonasfluorescens Loitokitok, having the following ATCC designations: [NCIB8286]; NRRL B-1244; NCIB 8865 strain COI; NCIB 8866 strain CO2; 1291[ATCC 17458; IFO 15837; NCIB 8917; LA; NRRL B-1864; pyrrolidine; PW2[ICMP 3966; NCPPB 967; NRRL B-899]; 13475; NCTC 10038; NRRL B-1603 [6;IFO 15840]; 52-1C; CCEB 488-A [BU 140]; CCEB 553 [IEM 15/47]; IAM 1008[AHH-27]; IAM 1055 [AHH-23]; 1 [IFO 15842]; 12 [ATCC 25323; NIH 11; denDooren de Jong 216]; 18 [IFO 15833; WRRL P-7]; 93 [TR-10]; 108[52-22;IFO 15832]; 143 [IFO 15836; PL]; 149 [2-40-40; IFO 15838]; 182 [IFO3081; PJ 73]; 184 [IFO 15830]; 185[W2 L-1]; 186 [IFO 15829; PJ 79]; 187[NCPPB 263]; 188 [NCPPB 316]; 189 [PJ227; 1208]; 191 [IFO 15834; PJ 236;22/1]; 194 [Klinge R-60; PJ 253]; 196 [PJ 288]; 197 [PJ 290]; 198[PJ302]; 201 [PJ 368]; 202 [PJ 372]; 203 [PJ 376]; 204 [IFO 15835; PJ 682];205[PJ686]; 206 [PJ 692]; 207 [PJ 693]; 208 [PJ 722]; 212 [PJ 832]; 215[PJ 849]; 216 [PJ885]; 267 [B-9]; 271 [B-1612]; 401 [C71A; IFO 15831; PJ187]; NRRL B-3178 [4; IFO 15841]; KY8521; 3081; 30-21; [IFO 3081]; N;PYR; PW; D946-B83 [BU 2183; FERM-P 3328]; P-2563 [FERM-P 2894; IFO13658]; IAM-1126 [43F]; M-1; A506 [A5-06]; A505[A5-05-1 ]; A526 [A5-26];B69; 72; NRRL B4290; PMW6 [NCIB 11615]; SC 12936; Al [IFO 15839]; F 1847[CDC-EB]; F 1848 [CDC 93]; NCIB 10586; P17; F-12; AmMS 257; PRA25;6133D02; 6519E01; Ni; SC15208; BNL-WVC; NCTC 2583 [NCIB 8194]; H13; 1013[ATCC 11251; CCEB 295]; IFO 3903; 1062; or Pf-5.

II. Auxotrophic Selection Markers

The present invention provides Pseudomonads and related cells that havebeen genetically modified to induce auxotrophy for at least onemetabolite. The genetic modification can be to a gene or genes encodingan enzyme that is operative in a metabolic pathway, such as an anabolicbiosynthetic pathway or catabolic utilization pathway. Preferably, thehost cell has all operative genes encoding a given biocatalytic activitydeleted or inactivated in order to ensure removal of the biocatalyticactivity. In a particular embodiment, the Pseudomonad is a Pseudomonasfluorescens cell.

One or more than one metabolic activity may be selected for knock-out orreplacement. In the case of native auxotrophy(ies), additional metabolicknockouts or replacements can be provided. Where multiple activities areselected, the auxotrophy-restoring selection markers can be of abiosynthetic-type (anabolic), of a utilization-type (catabolic), or maybe chosen from both types. For example, one or more than one activity ina given biosynthetic pathway for the selected compound may beknocked-out; or more than one activity, each from different biosyntheticpathways, may be knocked-out. The corresponding activity or activitiesare then provided by at least one recombinant vector which, upontransformation into the cell, restores prototrophy to the cell.

Compounds and molecules whose biosynthesis or utilization can betargeted to produce auxotrophic host cells include: lipids, including,for example, fatty acids; mono- and disaccharides and substitutedderivatives thereof, including, for example, glucose, fructose, sucrose,glucose-6-phosphate, and glucuronic acid, as well as Entner-Doudoroffand Pentose Phosphate pathway intermediates and products; nucleosides,nucleotides, dinucleotides, including, for example, ATP, dCTP, FMN, FAD,NAD, NADP, nitrogenous bases, including, for example, pyridines,purines, pyrimidines, pterins, and hydro-, dehydro-, and/or substitutednitrogenous base derivatives, such as cofactors, for example, biotin,cobamamide, riboflavine, thiamine; organic acids and glycolysis andcitric acid cycle intermediates and products, including, for example,hydroxyacids and amino acids; storage carbohydrates and storagepoly(hydroxyalkanoate) polymers, including, for example, cellulose,starch, amylose, amylopectin, glycogen, poly-hydroxybutyrate, andpolylactate.

In one embodiment, the biocatalytic activity(ies) knocked out to producethe auxotrophic host cell is selected from the group consisting of: thelipids; the nucleosides, nucleotides, dinucleotides, nitrogenous bases,and nitrogenous base derivatives; and the organic acids and glycolysisand citric acid cycle intermediates and products. Preferably, thebiocatalytic activity(ies) knocked out is selected from the groupconsisting of: the nucleosides, nucleotides, dinucleotides, nitrogenousbases, and nitrogenous base derivatives; and the organic acids andglycolysis and citric acid cycle intermediates and products. Morepreferably, the biocatalytic activity(ies) knocked out is selected fromthe group consisting of: the pyrimidine nucleosides, nucleotides,dinucleotides, nitrogenous bases, and nitrogenous base derivatives; andthe amino acids.

A given transgenic host cell may use one or more than one selectionmarker or selection marker system. For example, one or more biosynthesisselection marker(s) or selection marker system(s) according to thepresent invention may be used together with each other, and/or may beused in combination with a utilization-type selection marker orselection marker system according to the present invention. In any oneof these prototrophy-enabling embodiments, the host cell may alsocontain one or more non-auxotrophic selection marker(s) or selectionmarker system(s). Examples of non-auxotrophic selection marker(s) andsystem(s) include, for example: toxin-resistance marker genes such asantibiotic-resistance genes that encode an enzymatic activity thatdegrades an antibiotic; toxin-resistant marker genes, such as, forexample, imidazolinone-resistant mutants of acetolactate synthase(“ALS;” EC 2.2.1.6) in which mutation(s) are expressed that make theenzyme insensitive to toxin-inhibition exhibited by versions of theenzyme that do not contain such mutation(s). The compound(s) may exertthis effect directly; or the compound(s) may exert this effectindirectly, for example, as a result of metabolic action of the cellthat converts the compound(s) into toxin form or as a result ofcombination of the compound(s) with at least one further compound(s).

Bacterial-host-operative genes encoding such marker enzymes can beobtained from the bacterial host cell strain chosen for construction ofthe knock-out cell, from other bacteria, or from other organisms, andmay be used in native form or modified (e.g., mutated or sequencerecombined) form. For example, a DNA coding sequence for an enzymeexhibiting the knocked out biocatalytic activity may be obtained fromone or more organisms and then operatively attached to DNA regulatoryelements operative within the host cell. In specific, all of the chosenhost's intracellular genes that encode a selected enzymatic activity areknocked-out; the bacterial knock-out host is then transformed with avector containing at least one operative copy of a native or non-nativegene encoding an enzyme exhibiting the activity lost by the bacterialknockout.

Bacterial and other genes encoding such enzymes can be selected andobtained through various resources available to one of ordinary skill inthe art. These include the nucleotide sequences of enzyme codingsequences and species-operative DNA regulatory elements. Useful on-lineInterNet resources include, e.g.,: (1) the ExPASy proteomics facility(see the ENZYME and BIOCHEMICAL PATHWAYS MAPS features) of the SwissInstitute of Bioinformatics (Bâatiment Ecole de Pharmacie, Room 3041;Universitéde Lausanne; 1015 Lausanne-Dorigny; Switzerland) available at,e.g., http://us.expasy.org/; and (2) the GenBank facility and otherEntrez resources (see the PUBMED, PROTEIN, NUCLEOTIDE, STRUCTURE,GENOME, et al. features) offered by the National Center forBiotechnology Information (NCBI, National Library of Medicine, NationalInstitutes of Health, U.S. Dept. of Health & Human Services; Building38A; Bethesda, Md., USA) and available athttp://www.ncbi.nlm.nih.gov/entrez/guery.fcgi.

The selected coding sequence may be modified by altering the geneticcode thereof to match that employed by the bacterial host cell, and thecodon sequence thereof may be enhanced to better approximate thatemployed by the host. Genetic code selection and codon frequencyenhancement may be performed according to any of the various methodsknown to one of ordinary skill in the art, e.g.,oligonucleotide-directed mutagenesis. Useful on-line InterNet resourcesto assist in this process include, e.g.: (1) the Codon Usage Database ofthe Kazusa DNA Research Institute (2-6-7 Kazusa-kamatari, Kisarazu,Chiba 292-0818 Japan) and available at http://www.kazusa.or.jp/codon/;and (2) the Genetic Codes tables available from the NCBI Taxonomydatabase athttp://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c. Forexample, Pseudomonas species are reported as utilizing Genetic CodeTranslation Table 11 of the NCBI Taxonomy site, and at the Kazusa siteas exhibiting the codon usage frequency of the table shown athttp://www.kazusa.or.jp/codon/cgibin/.

In a particular embodiment, Pseudomonas fluorescens can be used as thehost cell. In one embodiment, Pseudomonas fluorescens provides at leastone auxotrophic selection marker gene. In an alternative embodiment,Pseudomonas fluorescens provides all auxotrophic selection marker genes.In a particular embodiment, Pseudomonas fluorescens can both be the hostcell and provide at least one, and preferably all, auxotrophic selectionmarker genes.

Biosynthetic Nucleoside and Nitrogenous Base Selection Markers

In one embodiment, a biosynthetic enzyme involved in anabolic metabolismcan be chosen as the auxotrophic selection marker. In particular, thebiosynthetic enzyme can be selected from those involved in biosynthesisof the nucleosides, nucleotides, dinucleotides, nitrogenous bases, andnitrogenous base derivatives.

In a particular embodiment at least one purine-type biosynthetic enzymecan be chosen as an auxotrophic selection marker. Such purinebiosynthetic enzymes include, for example, adeninephosphoribosyltransferases, adenylosuccinate lyases, adenylosuccinatesynthases, GMP synthases, IMP cyclohydrolases, IMP dehydrogenases,phosphoribosylamine-glycine ligases,phosphoribosyl-aminoimidazolecarboxamide formyltransferases,phosphoribosylaminoimidazole carboxylases, phosphoribosylaminoimidazolesuccinocarboxamide synthases,phosphoribosyl-formylglycinamidine cyclo ligases,phosphoribosyl-formylglycinamidine synthases, phosphoribosyl-glycinamideformyltransferases, ribose-phosphate diphosphokinases, andribose-5-phosphate-ammonia ligases.

In another particular embodiment, a pyrimidine-type biosynthetic enzymecan be chosen as an auxotrophic selection marker. Such pyrimidine-typebiosynthetic include enzymes involved in biosynthesis of UMP, such ascarbamate kinase (EC 2.7.2.2), carbamoyl-phosphate synthase (EC6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2), dihydroorotase(EC 3.5.2.3), dihydroorotate dehydrogenase (EC 1.3.3.1), orotatephosphoribosyltransferase (“OPRT;” EC 2.4.2.10), andorotidine-5′-phosphate decarboxylase (“ODCase;” EC 4.1.1.23).

Examples of genes encoding pyrimidine-type biosynthetic enzymes are wellknown. In the case of bacterial synthesis of UMP, examples of usefulgenes include: arcC genes, encoding carbamate kinases; carA and carBgenes, collectively encoding carbamoyl-phosphate synthases; pyrB genes,encoding aspartate carbamoytransferases; pyrC genes, encodingdihydroorotases; pyrD genes, singly or collectively encodingdihydroorotate dehydrogenases; pyrE genes encoding orotatephosphoribosyltransferases; and pyrF genes, encodingorotidine-5′-phosphate decarboxylases.

In a particular embodiment, an expression system according to thepresent invention will utilize a pyrF auxotrophic selection marker gene.pyrF genes encode ODCase, an enzyme required for the bacterialpyrimidine nucleotide biosynthesis pathway, by which the cell performsde novo synthesis of pyrimidine nucleotides proper (UTP, CTP), as wellas pyrimidine deoxynucleotides (dTTP, dCTP). The pathway's initialreactants are ATP, an amino group source (i.e. ammonium ion orL-glutamine), and a carboxyl group source (i.e. carbon dioxide orbicarbonate ion); the pathway's ultimate product is dTTP, with dCTP,UTP, and CTP also being formed in the process. Specifically, thebacterial de novo pyrimidine nucleotide biosynthesis pathway begins withthe formation of carbamoyl phosphate. Carbamoyl phosphate is synthesizedeither: (a) by action of carbamate kinase (EC 2.7.2.2), encoded by thearcC gene; or, more commonly, (b) by action of theglutamine-hydrolyzing, carbamoyl-phosphate synthase (EC 6.3.5.5), whosesmall and large subunits are encoded by the carA and carB genes,respectively. Carbamoyl phosphate is then converted to UDP by thefollowing six-step route: 1) conversion of carbamoyl phosphate toN-carbamoyl-L-aspartate, by aspartate carbamoyltransferase (EC 2.1.3.2),encoded by pyrB; then 2) conversion thereof to (S)-dihydroorotate, bydihydroorotase (EC 3.5.2.3), encoded by pyrC; then 3) conversion thereofto orotate, by dihydroorotate dehydrogenase (EC 1.3.3.1), encoded bypyrD gene(s); then 4) conversion thereof to orotidine-5′-monophosphate(“OMP”), by orotate phosphoribosyltransferase (“OPRT;” EC 2.4.2.10),encoded by pyrE; and then 5) conversion thereof touridine-5′-monophosphate (“UMP”), by orotidine-5′-phosphatedecarboxylase (“ODCase;” EC 4.1.1.23), encoded by pyrF. The UMP is thenutilized by a variety of pathways for synthesis of pyrimidinenucleotides (UTP, CTP, dTTP, dCTP), nucleic acids, nucleoproteins, andother cellular metabolites.

In bacteria in which one or more of the carA, carB, or pyrB-pyrF geneshas become inactivated or lost, or mutated to encode a non-functionalenzyme, the cell can still thrive if uracil is added to the medium,provided that the cell contains a functioning uracil salvage pathway.Most bacteria contain a native uracil salvage pathway, including thePseudomonads and related species. In a uracil salvage pathway, the cellimports and converts exogenous uracil into UMP, to synthesize therequired pyrimidine nucleotides. In this, uracil is reacted with5-phosphoribosyl-1-pyrophosphate to form UMP, by the action of eitheruracil phosphoribosyltransferase (EC 2.4.2.9), encoded by the upp gene,or by the bifunctional, pyrimidine operon regulatory protein (“pyrRbifunctional protein”), encoded by pyrR. The resulting UMP is thenconverted to UDP, and then the subsequent pyrimidine nucleotides, asdescribed above.

Consequently, a pyrF(−) Pseudomonad or related cell can be maintained onuracil-containing medium. After a pyrF gene-containing DNA construct istransfected into the pyrF(−) cell and expressed to form a functioningODCase enzyme, the resulting combined pyrF(+) plasmid-host cell systemcan be maintained in a medium lacking uracil.

The coding sequence of the pyrF gene for use in a Pseudomonad or relatedhost cell can be provided by any gene encoding an orotidine-5′-phosphatedecarboxylase enzyme (“ODCase”), provided that the coding sequence canbe transcribed, translated, and otherwise processed by the selectedPseudomonad or related host cell to form a functioning ODCase. The pyrFcoding sequence may be a native sequence, or it may be an engineeredsequence resulting from, for example, application of one or moresequence-altering, sequence-combining, and/or sequence-generatingtechniques known in the art. Before use as part of a pyrF selectionmarker gene, the selected coding sequence may first be improved oroptimized in accordance with the genetic code and/or the codon usagefrequency of a selected Pseudomonad or related host cell. Expressiblecoding sequences will be operatively attached to a transcriptionpromoter capable of functioning in the chosen host cell, as well as allother required transcription and translation regulatory elements. Anative coding sequence for a pyrF gene as described above may beobtained from a bacterium or from any other organism, provided that itmeets the above-described requirements.

In one embodiment, the pyrF coding sequence is isolated from thePseudomonad or related host cell in which it is intended to be used as aselection marker. The entire pyrF gene (including the coding sequenceand surrounding regulatory regions) can be isolated there from. In aparticular embodiment, a bacterium providing the pyrF gene or codingsequence will be selected from the group consisting of a member of theorder Pseudomonadales, a member of the suborder Pseudomonadineae, amember of the family Pseudomonadaceae, a member of the tribePseudomonadeae, a member of the genus Pseudomonas, and a member of thePseudomonas fluorescens species group (i.e. the “fluorescentpseudomonads”). In a particular embodiment, the bacterium will belong tothe species, Pseudomonas fluorescens.

In a particular embodiment, the pyrF gene contains the nucleic acidsequence of SEQ ID NO. 1 (Table 2), or a variant thereof. Alternatively,the ODCase encoded by the pyrF gene contains the amino acid sequence ofSEQ ID NO. 2 (Table 3), a variant thereof, or a variant having a codonsequence redundant therewith, in accordance with the genetic code usedby a given host cell according to the present invention.

Alternatively, the pyrF gene contains a nucleic acid sequence encodingan ODCase enzyme selected from the group consisting of a nucleic acidsequence at least 70%, 75%, 80%, 85%, 88%, 90%, and 95% homologous toSEQ ID No. 1. Likewise, the pyrF gene encodes an ODCase selected fromthe group consisting of an amino acid sequence at least 70%, 75%, 80%,85%, 88%, 90%, and 95% homologous to SEQ ID No. 2.

In another embodiment, the pyrF gene can contain a coding sequencehaving a nucleotide sequence at least 90%, 93%, 95%, 96%, 97%, 98% or99% homologous to the nucleotide sequence of nucleotides 974-1669 of SEQID NO: 1.

In a particular embodiment, the pyrF gene can contain a coding sequencehaving a codon sequence that hybridizes to the anti-codon sequence ofSEQ ID NO:3 (Table 4), when hybridization has been performed underhighly stringent hybridization conditions, or can have a codon sequenceredundant therewith. In a particularly particular embodiment, the pyrFgene will contain the nucleotide sequence of SEQ ID No. 3 TABLE 2PSEUDOMONAS FLUORESCENS PYRF NUCLEIC ACID SEQUENCEgatcagttgcggagccttggggtcatcccccagtttctgacgcaggcgcgacaccagcaagtcgatgctgcggtcgaSEQ ID NO. 1aagcctcgatggaacgcccacgggccgcgtccagcagctgttcgcggctcagcacacgccgcgggcgttcgataaacacccacaacaaacgaaactcggcgttggacagcggcaccaccaggccgtcatcggccaccagctggcgcagtacgctgttcaggcgccaagtgtcgaaacggatattggcccgctgttcggtgcggtcatcacgcacccggcgcaggatggtctggatacgcgcgaccagttcccggggttcgaacggcttggacatatagtcgtctgcccccagttccaggccgatgatgcggtcggtgggttcgcagcgggcggtgagcatcaggatcggaatgtccgattcggcgcgcagccagcggcacaatgtcagcccgtcttcgcccggcagcatcaggtcgagcaccaccacatcgaaggtctccgcttgcatggcctggcgcatggcgatgccgtcggtgacgcctgaggcgagaatattgaagcgtgccaggtagtcgatcagcagttcgcggatcggcacgtcgtcgtcgacaatcagcgcgcgggtgttccagcgcttgtcttcggcgatcaccgcgtcttttggcgcttcgtttacagggtcgcaaggggtatgcatagcgaggtcatctgcctggttgtggctgtcagcataggcgcccagttccagggctggaagtgctgggcgggcggtcatgtgcgcgaggctagccgggcggcgtattgggggcgtgtcgtgaatgtatcgggcttgaaacaattgccttgaatcgccggtattgggcgcttgatcggtatttaccgatcatcggatcccgcaacggcgctgcttgcgctacaatccgcgccgatttcgacttgcctgagagcccattccaatgtccgtctgccagactcctatcatcgtcgccctggattaccccacccgtgacgccgcactgaagctggctgaccagttggaccccaagctttgccgggtcaaggtcggcaaggaattgttcaccagttgcgcggcggaaatcgtcggcaccctgcgggacaaaggcttcgaagtgttcctcgacctcaaattccatgacatccccaacaccacggcgatggccgtcaaagccgcggccgagatgggcgtgtggatggtcaatgtgcactgctccggtggcctgcgcatgatgagcgcctgccgcgaagtgctggaacagcgcagcggccccaaaccgttgttgatcggcgtgaccgtgctcaccagcatggagcgcgaagacctggcgggcattggcctggatatcgagccgcaggtgcaagtgttgcgcctggcagccctggcgcagaaagccggcctcgacggcctggtgtgctcagccctggaagcccaggccctgaaaaacgcacatccgtcgctgcaactggtgacaccgggtatccgtcctaccggcagcgcccaggatgaccagcgccgtatcctgaccccgcgccaggccctggatgcgggctctgactacctggtgatcggccggccgatcagccaggcggcggatcctgcaaaagcgttggcagcggtcgtcgccgagatcgcctgatttttagagtgagcaaaaaatgtgggagctggcttgcctgcgatagtatcaactcggtatcacttagaaaccgagttgcttgcatcgcaggcaagccagctcccacatttgtttttgtggtgtgtcagctgactttgagcaccaacttcccgaagttctcgccgttgaacagcttcatcagcgtttccgggaatgtctccagcccttcgacaatatcttccttgctcttgagcttgccctgggccatccagccggccatttcctgacccgccgccgcgaagttcgccgcgtggtccatcaccacaaagccttccatacgcgcacggttgaccagcaatgacaggtagttcgccgggcctttgaccgcttccttgttgttgtactggctgattgcaccgcaaatcaccacgcgggctttgagcgccaggcggctgagcaccgcgtcgagaatatcgccgccgacgttatcgaaatacacgtccacgcctttggggcactcgcgcttgagggcggcgggcacgtcttcgcttttgtagtcgatggcggcgtcgaagcccagctcatcgaccaggaacttgcacttctcggcgccaccggcgatccccactacgcgacagcctttgagcttagcgatctgcccggcgatgctgcccacggcaccggcggcgccggagatcaccacggtgtcaccggctttcggtgcgccggtctccagcagagcaaagtaggccgtcatgccggtcatgcccagggcggacaggtagcggggcaggggcgccagcttggggtccaccttatagaaaccacggggctcgccaaggaagtaatcctgcacgcccagtgcaccgttcacgtagtcccccaccgcgaagttcggatggttcgaggcaagcaccttgcctacgcccagggcgcgcatcacttcgccgatgcctaccggtgggatgtaggacttgccttcattcatccagccacgca

TABLE 3 PSEUDOMONAS FLUORESCENS ODCASE AMINO ACID SEQUENCE Met Ser ValCys Gln Thr Pro Ile Ile Val Ala Leu Asp Tyr Pro Thr Arg Asp SEQ ID NO. 2Ala Ala Leu Lys Leu Ala Asp Gln Leu Asp Pro Lys Leu Cys Arg Val Lys ValGly Lys Glu Leu Phe Thr Ser Cys Ala Ala Glu Ile Val Gly Thr Leu Arg AspLys Gly Phe Glu Val Phe Leu Asp Leu Lys Phe His Asp Ile Pro Asn Thr ThrAla Met Ala Val Lys Ala Ala Ala Glu Met Gly Val Trp Met Val Asn Val HisCys Ser Gly Gly Leu Arg Met Met Ser Ala Cys Arg Glu Val Leu Glu Gln ArgSer Gly Pro Lys Pro Leu Leu Ile Gly Val Thr Val Leu Thr Ser Met Glu ArgGlu Asp Leu Ala Gly Ile Gly Leu Asp Ile Glu Pro Gln Val Gln Val Leu ArgLeu Ala Ala Leu Ala Gln Lys Ala Gly Leu Asp Gly Leu Val Cys Ser Ala LeuGlu Ala Gln Ala Leu Lys Asn Ala His Pro Ser Leu Gln Leu Val Thr Pro GlyIle Arg Pro Thr Gly Ser Ala Gln Asp Asp Gln Arg Arg Ile Leu Thr Pro ArgGln Ala Leu Asp Ala Gly Ser Asp Tyr Leu Val Ile Gly Arg Pro Ile Ser GlnAla Ala Asp Pro Ala Lys Ala Leu Ala Ala Val Val Ala Glu Ile Ala

TABLE 4 PSEUDOMONAS FLUORESCENS PYRF NUCLEIC ACID SEQUENCEatgtccgtctgccagactcctatcatcgtcgccctggattaccccacccgtgacgccgcactgaag SEQ.ID No. 3ctggctgaccagttggaccccaagctttgccgggtcaaggtcggcaaggaattgttcaccagttgcgcggcggaaatcgtcggcaccctgcgggacaaaggcttcgaagtgttcctcgacctcaaattccatgacatccccaacaccacggcgatggccgtcaaagccgcggccgagatgggcgtgtggatggtcaatgtgcactgctccggtggcctgcgcatgatgagcgcctgccgcgaagtgctggaacagcgcagcggccccaaaccgttgttgatcggcgtgaccgtgctcaccagcatggagcgcgaagacctggcgggcattggcctggatatcgagccgcaggtgcaagtgttgcgcctggcagccctggcgcagaaagccggcctcgacggcctggtgtgctcagccctggaagcccaggccctgaaaaacgcacatccgtcgctgcaactggtgacaccgggtatccgtcctaccggcagcgcccaggatgaccagcgccgtatcctgaccccgcgccaggccctggatgcgggctctgactacctggtgatcggccggccgatcagccaggcggcggatcctgcaaaagcgttggcagcggtcgtcgccgagatcgcc

In an alternate embodiment, an expression system according to thepresent invention will utilize a thyA auxotrophic selection marker gene.thyA genes encode thymidylate synthase (EC 2.1.1.45), an enzyme requiredfor the bacterial pyrimidine nucleotide biosynthesis pathway. Since DNAcontains thymine (5-methyluracil) as a major base instead of uracil, thesynthesis of thymidine monophospate (dTMP or thymidylate) is essentialto provide dTTP (thymidine triphosphate) needed for DNA replicationtogether with dATP, dGTP, and dCTP. Methylation of dUMP by thymidylatesynthase utilizing 5,10-methylenetetrahydrofolate as the source of themethyl group generates thymidylate. Thymidylate synthesis can beinterrupted, and consequently the synthesis of DNA arrested, by theremoval, inhibition, or disruption of thymidylate synthase.

In bacteria in which the thyA gene has become inactivated or lost, ormutated to encode a non-functional enzyme, the cell can still thrive ifexogenous thymidine is added to the medium.

In Pseudomonas fluorescens, the addition of an E.coli tdk gene, encodingthymidine kinase, is required for survival on exogenous thymidine.Therefore, prior to selection, a plasmid comprising a tdk gene can beused to transform thyA(−) P. fluorescens host cells, generating athyA(−)/ptdk cell, allowing survival on a thymidine containing medium.Alternatively, a tdk gene producing a functional thymidylate synthaseenzyme capable of utilizing exogenous thymidine in Pseudomonasfluorescens can be inserted into the genome, producing a thyA(−)/tdk(+)host cell. After a thyA gene-containing DNA construct is transfectedinto the thyA(−)/ptdk cell and expressed to form a functioningthymidylate synthase enzyme, the resulting combined thyA(+) plasmid-hostcell system can be maintained in a medium lacking thymidine.

The coding sequence of the thyA gene for use in a Pseudomonad or relatedhost cell can be provided by any gene encoding a thymidylate synthaseenzyme (“TS”), provided that the coding sequence can be transcribed,translated, and otherwise processed by the selected Pseudomonad orrelated host cell to form a functioning TS. The thyA coding sequence maybe a native sequence, or it may be an engineered sequence resultingfrom, for example, application of one or more sequence-altering,sequence-combining, and/or sequence-generating techniques known in theart. Before use as part of a thyA selection marker gene, the selectedcoding sequence may first be improved or optimized in accordance withthe genetic code and/or the codon usage frequency of a selectedPseudomonad or related host cell. Expressible coding sequences will beoperatively attached to a transcription promoter capable of functioningin the chosen host cell, as well as all other required transcription andtranslation regulatory elements. A native coding sequence for a thyAgene as described above may be obtained from a bacterium or from anyother organism, provided that it meets the above-described requirements.

In one embodiment, the thyA coding sequence is isolated from thePseudomonad or related host cell in which it is intended to be used as aselection marker. The entire thyA gene (including the coding sequenceand surrounding regulatory regions) can be isolated there from. In aparticular embodiment, a bacterium providing the thyA gene or codingsequence will be selected from the group consisting of a member of theorder Pseudomonadales, a member of the suborder Pseudomonadineae, amember of the family Pseudomonadaceae, a member of the tribePseudomonadeae, a member of the genus Pseudomonas, and a member of thePseudomonas fluorescens species group (i.e. the “fluorescentpseudomonads”). In a particular embodiment, the bacterium will belong tothe species, Pseudomonas fluorescens.

In a particular embodiment, the thyA gene contains the nucleic acidsequence of SEQ ID NO. 4 (Table 5). Alternatively, the TS encoded by thethyA gene contains the amino acid sequence of SEQ ID NO. 5 (Table 6), avariant thereof, or a variant having a codon sequence redundanttherewith, in accordance with the genetic code used by a given host cellaccording to the present invention. TABLE 5 PSEUDOMONAS FLUORESCENS THYANUCLEIC ACID SEQUENCEatgaagcaatatctcgaactactgaacgacgtcgtgaccaatggattgaccaagggcgatcgcac SEQ IDNO. 4 cggcaccggcaccaaagccgtatttgcccgtcagtatcggcataacttggccgacggcttcccgctgctgaccaccaagaagcttcatttcaaaagtatcgccaacgagttgatctggatgttgagcggcaacaccaacatcaagtggctcaacgaaaatggcgtgaaaatctgggacgagtgggccaccgaagacggcgacctgggcccggtgtacggcgagcaatggaccgcctggccgaccaaggacggcggcaagatcaaccagatcgactacatggtccacaccctcaaaaccaaccccaacagccgccgcatcctgtttcatggctggaacgtcgagtacctgccggacgaaaccaagagcccgcaggagaacgcgcgcaacggcaagcaagccttgccgccgtgccatctgttgtaccaggcgttcgtgcatgacgggcatctgtcgatgcagttgtatatccgcagctccgacgtcttcctcggcctgccgtacaacaccgccgcgttggccttgctgactcacatgctggctcagcaatgcgacctgatccctcacgagatcatcgtcaccaccggcgacacccatgcttacagcaaccacatggaacagatccgcacccagctggcgcgtacgccgaaaaagctgccggaactggtgatcaagcgtaaacctgcgtcgatctacgattacaagtttgaagactttgaaatcgttggctacgacgccgacccgagcatcaaggctgacgtggctatctga

TABLE 6 PSEUDOMONAS FLUORESCENS TS AMINO ACID SEQUENCEMKQYLELLNDVVTNGLTKGDRTGTGTKAVFARQYRHNLADGFPLLTTKKLHFKSIANELIWMLSG SEQ IDNO. 5 NTNIKWLNENGVKIWDEWATEDGDLGPVYGEQWTAWPTKDGGKINQIDYMVHTLKTNPNSRRILFHGWNVEYLPDETKSPQENARNGKQALPPCHLLYQAFVHDGHLSMQLYIRSSDVFLGLPYNTAALALLTHMLAQQCDLIPHEIIVTTGDTHAYSNHMEQIRTQLARTPKKLPELVIKRKPASIYDYKFEDFEIVGYDADPSIKADVAI

Biosynthetic Amino Acid Selection Markers

In an alternative embodiment, the biosynthetic enzyme involved inanabolic metabolism chosen as the auxotrophic selection marker can beselected from those involved in the biosynthesis of amino acids. Inparticular embodiments, the biosynthetic amino acid enzymes are selectedfrom the group consisting of enzymes active in the biosynthesis of: theGlutamate Family (Glu; Gln, Pro, and Arg); the Aspartate Family (Asp;Asn, Met, Thr, Lys, and Ile); the Serine Family (Ser; Gly and Cys); thePyruvate Family (Ala, Val, and Leu); the Aromatic Family (Trp, Phe, andTyr); and the Histidine Family (His). Examples of genes and enzymesinvolved in these biosynthetic pathways include: the Glutamate Familymember arg, gdh, gln, and, pro genes, including, for example, argA-argH,gdhA, glnA, proA, proC; the Aspartate Family member asd, asn, asp, dap,lys, met, and thr genes, including, for example, asnA, asnB, aspC, dapA,dapB, dapD-dapF, lysA, lysC, metA-metC, metE, metH, metL, thrA-thrC; theSerine Family member cys, gly, and ser genes, including, for example,cysE, cysK, glyA, serA-serC; the Aromatic Family member aro, phe, trp,and tyr genes, including, for example, aroA-aroH, aroK, aroL, trpAtrpE,tyrA, and tyrB; and the Histidine Family member his genes, includinghisA-hisD, hisF-hisH.

In a further particular embodiment, the auxotrophic selection marker canbe selected from enzymes involved in the biosynthesis of members of theGlutamate Family. Examples of useful Glutamate Family auxotrophicselection markers include the following, listed with representativeexamples of their encoding genes: argA, encoding N-acetylglutamatesynthases, amino acid acetyltransferases; argB, encoding acetylglutamatekinases; argC, encoding N-acetyl-gammaglutamylphosphate reductases;argD, encoding acetylornithine delta-aminotransferases; argE, encodingacetylornithine deacetylases; argF and argI, encoding ornithinecarbamoyltransferases; argG, encoding argininosuccinate synthetases;argH, encoding argininosuccinate lyases; gdhA, encoding glutamatedehydrogenases; glnA, encoding glutamine synthetases; proA, encodinggamma-glutamylphosphate reductases; proB, encoding gamma-glutamatekinases; and proC, encoding pyrroline-5-carboxylate reductases.

In one embodiment, an amino acid biosynthesis selection marker gene canbe at least one member of the proline biosynthesis family, in particularproA, proB, or proC. In a particular embodiment, the prolinebiosynthesis selection marker gene can comprise a proC gene. proC genesencode an enzyme catalyzing the final step of the proline biosynthesispathway. In bacteria, the proline (i.e. L-proline) biosynthesis pathwaycomprises a three-enzyme process, beginning with L-glutamic acid. Thesteps of this process are: 1) conversion of L-glutamic acid toL-glutamyl-5-phosphate, by glutamate-5-kinase (“GK;” EC 2.7.2.11),encoded by proB; then 2a) conversion thereof toL-glutamate-5-semialdehyde, by glutamate-5-semialdehyde dehydrogenase(EC 1.2.1.41), also known as glutamyl-5-phosphate reductase (“GPR”),encoded by proA, followed by 2b) spontaneous cyclization thereof to form.1-pyrroline-5-carboxylate; and then 3) conversion thereof to L-proline,by Δ¹-pyrroline-5-carboxylate reductase (“P5CR;” EC 1.5.1.2), encoded byproC. In most bacteria, proC encodes the P5CR subunit, with the activeP5CR enzyme being a homo-multimer thereof.

In bacteria in which one or more of the proA, proB, or proC genes hasbecome inactivated or lost, or mutated to encode a non-functionalenzyme, the cell can still thrive if proline is added to the medium.Consequently, a proC(−) Pseudomonad or related cell can be maintained ona proline-containing medium. After a proC gene-containing DNA constructis transfected into the proC(−) cell and expressed to form a functioningP5CR enzyme, the resulting combined proC(+) plasmid-host cell system canbe maintained in a medium lacking proline.

The coding sequence of the proC gene for use in a Pseudomonad or relatedhost cell can be provided by any gene encoding anΔ¹-pyrroline-5-carboxylate reductase enzyme (P5CR), provided that thecoding sequence can be transcribed, translated, and otherwise processedby the selected Pseudomonad or related host cell to form a functioningP5CR. The proC coding sequence may be a native sequence, or it may be anengineered sequence resulting from, for example, application of one ormore sequence-altering, sequence-combining, and/or sequence-generatingtechniques known in the art. Before use as part of a proC selectionmarker gene, the selected coding sequence may first be improved oroptimized in accordance with the genetic code and/or the codon usagefrequency of a selected Pseudomonad or related host cell. Expressiblecoding sequences will be operatively attached to a transcriptionpromoter capable of functioning in the chosen host cell, as well as allother required transcription and translation regulatory elements. Anative coding sequence for a proC gene as described above may beobtained from a bacterium or from any other organism, provided that itmeets the above-described requirements.

In one embodiment, the proC coding sequence is isolated from thePseudomonad or related host cell in which it is intended to be used as aselection marker. The entire proC gene (including the coding sequenceand surrounding regulatory regions) can be isolated therefrom. In aparticular embodiment, a bacterium providing the proC gene or codingsequence will be selected from the group consisting of a member of theorder Pseudomonadales, a member of the suborder Pseudomonadineae, amember of the family Pseudomonadaceae, a member of the tribePseudomonadeae, a member of the genus Pseudomonas, and a member of thePseudomonas fluorescens species group (i.e. the “fluorescentpseudomonads”). In a particular embodiment, the bacterium will belong tothe species, Pseudomonas fluorescens.

In a particular embodiment, the proC gene contains the nucleic acidsequence of SEQ ID NO. 6 (Table 7), or a variant thereof. Alternatively,the P5CR encoded by the proC gene contains the amino acid sequence ofSEQ ID NO. 7 (Table 8), a variant thereof, or a variant having a codonsequence redundant therewith, in accordance with the genetic code usedby a given host cell according to the present invention.

Alternatively, the proC gene contains a nucleic acid sequence encodingan P5CR enzyme that is at least 70%, 75%, 80%, 85%, 88%, 90%, and 95%homologous to SEQ ID No. 6. Likewise, the proC gene encodes an ODCasethat is at least 70%, 75%, 80%, 85%, 88%, 90%, and 95% homologous to SEQID No. 7.

In another embodiment, the proC gene can contain a coding sequence atleast 90%, 93%, 95%, 96%, 97%, 98% or 99% homologous to the nucleotidesequence of SEQ. ID NO. 8 (Table 9).

In a particular embodiment, the proC gene can contain a coding sequencehaving a codon sequence that hybridizes to the anti-codon sequence ofSEQ ID NO. 8, when hybridization has been performed under stringenthybridization conditions, or can have a codon sequence redundanttherewith. In a particularly particular embodiment, the proC gene willcontain the nucleotide sequence of SEQ ID NO. 8. TABLE 7 PSEUDOMONASFLUORESCENS PROC NUCLEIC ACID SEQUENCEgcccttgagttggcacttcatcggccccattcaatcgaacaagactcgtgccatcgccgagcacttcgcttggSEQ ID NO. 6gtgcactccgtggaccgcctgaaaatcgcacaacgcctgtccgaacaacgcccggccgacctgccgccgctcaatatctgcatccaggtcaatgtcagtggcgaagccagcaagtccggctgcacgcccgctgacctgccggccctggccacagcgatcagcgccctgccgcgcttgaagctgcggggcttgatggcgattcccgagccgacgcaagaccgggcggagcaggatgcggcgttcgccacggtgcgcgacttgcaagccagcttgaacctggcgctggacacactttccatgggcatgagccacgaccttgagtcggccattgcccaaggcgccacctgggtgcggatcggtaccgccctgtttggcgcccgcgactacggccagccgtgaaatggctgacatccctcgaaataaggacctgtcatgagcaacacgcgtattgcctttatcggcgccggtaacatggcggccagcctgatcggtggcctgcgggccaagggcctggacgccgagcagatccgcgccagcgaccccggtgccgaaacccgcgagcgcgtcagagccgaacacggtatccagaccttcgccgataacgccgaggccatccacggcgtcgatgtgatcgtgctggcggtcaagccccaggccatgaaggccgtgtgcgagagcctgagcccgagcctgcaaccccatcaactggtggtgtcgattgccgctggcatcacctgcgccagcatgaccaactggctcggtgcccagcccattgtgcgctgcatgcccaacaccccggcgctgctgcgccagggcgtcagcggtttgtatgccactggcgaagtcaccgcgcagcaacgtgaccaggcccaggaactgctgtctgcggtgggcatcgccgtgtggctggagcaggaacagcaactggatgcggtcaccgccgtctccggcagcggcccggcttacttcttcctgttgatcgaggccatgacggccgcaggcgtcaagctgggcctgccccacgacgtggccgagcaactggcggaacaaaccgccctgggcgccgccaagatggcggtcggcagcgaggtggatgccgccgaactgcgccgtcgcgtcacctcgccaggtggtaccacacaagcggctattgagtcgttccaggccgggggctttgaagccctggtggaaacagcactgggtgccgccgcacatcgttcagccgagatggctgagcaactgggcaaatagtcgtcccttaccaaggtaatcaaacatgctcggaatcaatgacgctgccattttcatcatccagaccctgggcagcctgtacctgctgatcgtactgatgcgctttatcctgcaactggtgcgtgcgaacttctacaacccgctgtgccagttcgtggtgaaggccacccaaccgctgctcaagccgctgcgccgggtgatcccgagcctgttcggcctggacatgtcgtcgctggtgctggcgctgttgctgcagattttgctgttcgtggtgatcctgatgctcaatggataccaggccttcaccgtgctgctgttgccatggggcctgatcgggattttctcgctgttcctgaagatcattttctggtcgatgatcatcagcgtgatcctgtcctgggtcgcaccgggtagccgtagcccgggtgccgaattggtggctcagatcaccgagccggtgctggcacccttccgtcgcctgattccgaacctgggtggcctggatatctcgccgatcttcgcgtttatc

TABLE 8 PSEUDOMONAS FLUORESCENS P5CR AMINO ACID SEQUENCE Met Ser Asn ThrArg Ile Ala Phe Ile Gly Ala Gly Asn Met Ala Ala Ser Leu SEQ ID NO. 7 IleGly Gly Leu Arg Ala Lys Gly Leu Asp Ala Glu Gln Ile Arg Ala Ser Asp ProGly Ala Glu Thr Arg Glu Arg Val Arg Ala Glu His Gly Ile Gln Thr Phe AlaAsp Asn Ala Glu Ala Ile His Gly Val Asp Val Ile Val Leu Ala Val Lys ProGln Ala Met Lys Ala Val Cys Glu Ser Leu Ser Pro Ser Leu Gln Pro His GlnLeu Val Val Ser Ile Ala Ala Gly Ile Thr Cys Ala Ser Met Thr Asn Trp LeuGly Ala Gln Pro Ile Val Arg Cys Met Pro Asn Thr Pro Ala Leu Leu Arg GlnGly Val Ser Gly Leu Tyr Ala Thr Gly Glu Val Thr Ala Gln Gln Arg Asp GlnAla Gln Glu Leu Leu Ser Ala Val Gly Ile Ala Val Trp Leu Glu Gln Glu GlnGln Leu Asp Ala Val Thr Ala Val Ser Gly Ser Gly Pro Ala Tyr Phe Phe LeuLeu Ile Glu Ala Met Thr Ala Ala Gly Val Lys Leu Gly Leu Pro His Asp ValAla Glu Gln Leu Ala Glu Gln Thr Ala Leu Gly Ala Ala Lys Met Ala Val GlySer Glu Val Asp Ala Ala Glu Leu Arg Arg Arg Val Thr Ser Pro Gly Gly ThrThr Gln Ala Ala Ile Glu Ser Phe Gln Ala Gly Gly Phe Glu Ala Leu Val GluThr Ala Leu Gly Ala Ala Ala His Arg Ser Ala Glu Met Ala Glu Gln Leu GlyLys

TABLE 9 PSEUDOMONAS FLUORESCENS PROC NUCLEIC ACID SEQUENCEatgagcaacacgcgtattgcctttatcggcgccggtaacatggcggccagcctgatcggtggc SEQ IDNO. 8 ctgcgggccaagggcctggacgccgagcagatccgcgccagcgaccccggtgccgaaacccgcgagcgcgtcagagccgaacacggtatccagaccttcgccgataacgccgaggccatccacggcgtcgatgtgatcgtgctggcggtcaagccccaggccatgaaggccgtgtgcgagagcctgagcccgagcctgcaaccccatcaactggtggtgtcgattgccgctggcatcacctgcgccagcatgaccaactggctcggtgcccagcccattgtgcgctgcatgcccaacaccccggcgctgctgcgccagggcgtcagcggtttgtatgccactggcgaagtcaccgcgcagcaacgtgaccaggcccaggaactgctgtctgcggtgggcatcgccgtgtggctggagcaggaacagcaactggatgcggtcaccgccgtctccggcagcggcccggcttacttcttcctgttgatcgaggccatgacggccgcaggcgtcaagctgggcctgccccacgacgtggccgagcaactggcggaacaaaccgccctgggcgccgccaagatggcggtcggcagcgaggtggatgccgccgaactgcgccgtcgcgtcacctcgccaggtggtaccacacaagcggctattgagtcgttccaggccgggggctttgaagccctggtggaaacagcactgggtgccgccgcacatcgttcagccgagatggctgagcaactgggcaaa

Utilization Selection Markers

In one embodiment, an enzyme involved in the catabolic utilization ofmetabolites can be chosen as the auxotrophic selection marker. Inparticular, the enzymes can be selected from those involved in theutilization of a carbon source. Examples of such enzymes include, forexample, sucrases, lactases, maltases, starch catabolic enzymes,glycogen catabolic enzymes, cellulases, andpoly(hydroxyalkanoate)depolymerases. If the bacterial host cell exhibitsnative catabolic activity of the selected type, it can be knocked-outbefore transformation with the prototrophy-restoring vector. Bacteriaexhibiting native auxotrophy for these compounds can also be used intheir native state for such transformation. In those embodiments inwhich a compound not importable or diffusible into the cell can beselected and supplied to the medium, the prototrophy restoring orprototrophy-enabling enzyme(s) can be secreted for use. In that case,the secreted enzyme(s) can degrade the compound extracellularly toproduce smaller compounds, for example glucose, that are diffusible orimportable into the cell, by selecting or designing the coding sequenceof the enzyme(s) to include a coding sequence for a secretion signalpeptide operative within the chosen host cell. In these embodiments, theprototrophy-restorative gene can be selected or be engineered to includea coding sequence for a secretion signal peptide operative within thechosen host cell to obtaining transport of the enzyme across thecytoplasmic membrane. In either of these embodiments, or those in whichthe selected compound is importable or diffusible into the cell, thecell will be grown in medium supplying no other carbon source apart fromthe selected compound.

In a carbon-source-utilization-based marker system, everyprototrophy-restorative or prototrophy-enabling carbon-sourceutilization enzyme can be involved in utilization of only one carbonsource. For example, two genes from the same catabolic pathway may beexpressed together on one vector or may be co-expressed separately ondifferent vectors in order to provide the prototrophy. Specific examplesof such multi-gene carbon-source-utilization-based marker systemsinclude, for example, the use of glycogen as the sole carbon source withtransgenic expression of both a glycogen phosphorylase and an(alpha-1,4)glucantransferase; and the use of starch as the sole carbonsource with transgenic expression of both an alpha-amylase, and analpha(1->6) glucosidase. However, the selected single- or multi-genecarbon-source marker system can be used simultaneously with other typesof marker system(s) in the same host cell, provided that the only carbonsource provided to the cell is the compound selected for use in thecarbon-source catabolic selection marker system.

Other examples of useful enzymes for biochemical-utilization-typeactivities are well known in the art, and can include racemases andepimerases that are capable of converting a non-utilizable D-carbonsource, supplied to the cell, to a nutritive L-carbon source. Examplesof these systems include, for example: a D-acid or a D-acyl compoundused with trangenic expression of the corresponding racemase; andlactate used with transgenically expressed lactate racemase.

Similarly, where an amino acid biosynthetic activity has been selectedfor use in the marker system, the auxotrophy may also be overcome bysupplying the cell with both a non-utilizable R-amino acid and anR-amino acid racemase or epimerase (EC 5.1.1) that converts the R-aminoacid into the corresponding L-amino acid for which the cell isauxotrophic.

Trait Stacking

A plurality of phenotypic changes can also be made to a host cell,before or after insertion of an auxotrophic selection marker gene, fortarget gene expression, according to the present invention. For example,the cell can be genetically engineered, either simultaneously orsequentially, to exhibit a variety of enhancing phenotypic traits. Thisprocess is referred to as “trait stacking.” A pryF deletion may bepresent as one such phenotypic trait. In such a strain, a pyrF gene,according to the present invention, can be used on a suicide vector asboth a selectable marker and a counterselectable marker (in the presenceof 5′-fluoroorotic acid) in order to effect a cross-in/cross-out alleleexchange of other desirable traits, Thus, a pyrF gene according to thepresent invention may be used in a process for “trait stacking” a hostcell. In such a process, a suicide vector containing such a pyrF genecan be transformed into the host cell strain in a plurality of separatetransformations; in each such procedure the re-establishment of the pyrFphenotype can be used to create, ad infinitum, subsequentgenetically-enhancing phenotypic change. Thus, not only can the pyrFgene itself provide a trait, it can be used to obtain additionalphenotypic traits in a process of trait-stacking.

In one embodiment, the present invention provides auxotrophicPseudomonads and related bacteria that have been further geneticallymodified to induce additional auxotrophies. For example, a pyrF(−)auxotroph can be further modified to inactivate another biosyntheticenzyme present in an anabolic or catabolic pathway, such as through theinactivation of a proC gene or a thyA gene. In this way, multipleauxotrophies in the host cell can be produced.

In another embodiment, genetic alterations can be made to the host cellin order to improve the expression of recombinant polypeptides in thehost cell. Further modifications can include genetic alterations thatallow for a more efficient utilization of a particular carbon source,thereby optimizing the overall efficiency of the entire fermentation.

In one particular embodiment, auxotrophic host cells are furthermodified by the insertion of a lacI containing transgene into the hostchromosome. Preferably, the lacI transgene, or derivate thereof, isother than part of a whole or truncated structural gene containingPlacI-lacI-lacZYA construct.

Modifications to Induce Auxotrophism

A Pseudomonad or related host cell selected for use in an expressionsystem according to the present invention can be deficient in itsability to express any functional biocatalyst exhibiting the selectedauxotrophic activity. For example, where an orotidine-5′-phosphatedecarboxylase activity is selected, the host cell can be deficient inits ability to express a) any pyrF gene product (i.e. any functionalODCase enzyme), and b) any effective replacement therefore (i.e. anyother biocatalyst having ODCase activity). In a one embodiment, the hostcell will be made biocatalytically-deficient for the selected activityby altering its genomic gene(s) so that the cell cannot express, fromits genome, a functional enzyme involved in the targeted auxotrophy(i.e. ODCase). In other words, the prototrophic cell (activity(+) cell)will become auxotrophic through the “knock-out” of a functionalenzymatic encoding gene involved in the targeted prototrophic pathway(i.e. an activity(−) cell). This alteration can be done by altering thecell's genomic coding sequence(s) of the gene(s) encoding the selectedactivty(ies). In one embodiment, the coding sequence alteration(s) willbe accomplished by introducing: insertion or deletion mutation(s) thatchange the coding sequence reading frame(s); substitution or inversionmutations that alter a sufficient number of codons; and/or deletionmutations that delete a sufficiently large group of contiguous codonsthere from capable of producing a non-functional enzyme.

In a one embodiment in which the host cell strain has also provided theauxotrophic gene(s) for use as selection marker(s) therein, preferablyeach of the selected gene's transcription promoter and/or transcriptionterminator element(s) can also be inactivated by introduction ofmutation(s), including deletion mutations. For example, thetranscription element inactivation can be optionally performed inaddition to the coding sequence alteration(s) described above. In a oneembodiment in which the host cell strain has also provided theauxotrophic selection marker gene(s), all of the selected gene(s)'s DNAcan be deleted from the host cell genome.

Such knock-out strains can be prepared according to any of the variousmethods known in the art as effective. For example, homologousrecombination vectors containing homologous targeted gene sequences 5′and 3′ of the desired nucleic acid deletion sequence can be transformedinto the host cell. Ideally, upon homologous recombination, a desiredtargeted enzymatic gene knock-out can be produced.

Specific examples of gene knock-out methodologies include, for example:Gene inactivation by insertion of a polynucleotide has been previouslydescribed. See, e.g., D L Roeder & A Collmer, Marker-exchangemutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi, JBacteriol. 164(1):51-56 (1985). Alternatively, transposon mutagenesisand selection for desired phenotype (such as the inability to metabolizebenzoate or anthranilate) can be used to isolate bacterial strains inwhich target genes have been insertionally inactivated. See, e.g., KNida & P P Cleary, Insertional inactivation of streptolysin S expressionin Streptococcus pyogenes, J Bacteriol. 155(3):1156-61 (1983). Specificmutations or deletions in a particular gene can be constructed usingcassette mutagenesis, for example, as described in J A Wells et al.,Cassette mutagenesis: an efficient method for generation of multiplemutations at defined sites, Gene 34(2-3):315-23 (1985); whereby director random mutations are made in a selected portion of a gene, and thenincorporated into the chromosomal copy of the gene by homologousrecombination.

In one embodiment, both the organism from which the selection markergene(s) is obtained and the host cell in which the selection markergene(s) is utilized can be selected from a prokaryote. In a particularembodiment, both the organism from which the selection marker gene(s) isobtained and the host cell in which a selection marker gene(s) isutilized can be selected from a bacteria. In another embodiment, boththe bacteria from which the selection marker gene(s) is obtained and thebacterial host cell in which a selection marker gene(s) is utilized,will be selected from the Proteobacteria. In still another embodiment,both the bacteria from which the selection marker gene(s) is obtainedand the bacterial host cells in which a selection marker gene(s) isutilized, can be selected from the Pseudomonads and closely relatedbacteria or from a Subgroup thereof, as defined below.

In a particular embodiment, both the selection marker gene(s) sourceorganism and the host cell can be selected from the same species.Preferably, the species will be a prokaryote; more preferably abacterium, still more preferably a Proteobacterium. In anotherparticular embodiment, both the selection marker gene(s) source organismand the host cell can be selected from the same species in a genusselected from the Pseudomonads and closely related bacteria or from aSubgroup thereof, as defined below. In one embodiment, both theselection marker gene(s) source organism and the host cell can beselected from a species of the genus Pseudomonas, particularly thespecies Pseudomonas fluorescens, and preferably the species Pseudomonasfluorescens biotype A.

III. LacI Insertion

The present invention provides Pseudomonads and related cells that havebeen genetically modified to contain a chromosomally insert lacItransgene or derivative, other than as part of a whole or truncatedPlacI-lacI-lacZYA operon. In one embodiment, the lacI insert providesstringent expression vector control through the expression of the LacIrepressor protein which binds to the lacO sequence or derivative on thevector, and inhibits a Plac-Ptac family promoter on the vector. Theresult is reduced basal levels of recombinant polypeptide expressionprior to induction.

In one embodiment, Pseudomonad host cells containing a chromosomalinsertion of a native E.coli lacI gene, or lacI gene derivative such aslacI^(Q) or lacI^(Q 1,) are provided wherein the lacI insert is otherthan part of a whole or truncated, structural gene-containingPlacI-lacI-lacZYA construct. Other derivative lacI transgenes useful inthe present invention include: lacI derivatives that have altered codonsequences different from a native lacI gene (for example, the nativeE.coli lacI gene contains a ‘gtg’ initiation codon, and this may bereplaced by an alternative initiation codon effective for translationinitiation in the selected expression host cell, e.g., ‘atg’); lacIderivatives that encode LacI proteins having mutated amino acidsequences, including temperature-sensitive lacI mutants, such as thatencoded by lacI^(ts) (or “lacI(Ts)”), which respond to a shift intemperature in order to achieve target gene induction, e.g., a shift upto 42° C. (see, e.g., Bukrinsky et al., Gene 70:415-17 (1989); N Hasan &W Szybalski, Gene 163(1):35-40 (1995); H Adari et al., DNA Cell Biol.14:945-50 (1995)); LacI mutants that respond to the presence ofalternative sugars other than lactose in order to achieve induction,e.g., arabinose, ribose, or galactose (see, e.g., WO 99/27108 for LacRepressor Proteins with Altered Responsivity); and LacI mutants thatexhibit at least wild-type binding to lac operators, but enhancedsensitivity to an inducer (e.g., IPTG), or that exhibit enhanced bindingto lac operators, but at least wild-type de-repressibility (see, e.g., LSwint-Kruse et al., Biochemistry 42(47):14004-16 (2003)).

In a particular embodiment, the gene encoding the Lac repressor proteininserted into the chromosome is identical to that of native E.coli lacIgene, and has the nucleic acid sequence of SEQ ID NO. 9 (Table 10). Inanother embodiment, the gene inserted into the host chromosome encodesthe Lac repressor protein having the amino acid sequence of SEQ ID NO.10 (Table 1 1). TABLE 10 NUCLEIC ACID SEQUENCE OF NATIVE E. COLI LACIGENE Gacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtca SEQID NO 9 attcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcgttttacaacgtcgtga

TABLE 11 AMINO ACID SEQUENCE OF LACI REPRESSOR Met Lys Pro Val Thr LeuTyr Asp Val Ala Glu Tyr Ala Gly Val SEQ ID NO. 10 Ser Tyr Gln Thr ValSer Arg Val Val Asn Gln Ala Ser His Val Ser Ala Lys Thr Arg Glu Lys ValGlu Ala Ala Met Ala Glu Leu Asn Tyr Ile Pro Asn Arg Val Ala Gln Gln LeuAla Gly Lys Gln Ser Leu Leu Ile Gly Val Ala Thr Ser Ser Leu Ala Leu HisAla Pro Ser Gln Ile Val Ala Ala Ile Lys Ser Arg Ala Asp Gln Leu Gly AlaSer Val Val Val Ser Met Val Glu Arg Ser Gly Val Glu Ala Cys Lys Ala AlaVal His Asn Leu Leu Ala Gln Arg Val Ser Gly Leu Ile Ile Asn Tyr Pro LeuAsp Asp Gln Asp Ala Ile Ala Val Glu Ala Ala Cys Thr Asn Val Pro Ala LeuPhe Leu Asp Val Ser Asp Gln Thr Pro Ile Asn Ser Ile Phe Ser His Glu AspGly Thr Arg Leu Gly Val Glu His Leu Val Ala Leu Gly His Gln Gln Ile AlaLeu Leu Ala Gly Pro Leu Ser Ser Val Ser Ala Arg Leu Arg Leu Ala Gly TrpHis Lys Tyr Leu Thr Arg Asn Gln Ile Gln Pro Ile Ala Glu Arg Glu Gly AspTrp Ser Ala Met Ser Gly Phe Gln Gln Thr Met Gln Met Leu Asn Glu Gly IleVal Pro Thr Ala Met Leu Val Ala Asn Asp Gln Met Ala Leu Gly Ala Met ArgAla Ile Thr Glu Ser Gly Leu Arg Val Gly Ala Asp Ile Ser Val Val Gly TyrAsp Asp Thr Glu Asp Ser Ser Cys Tyr Ile Pro Pro Ser Thr Thr Ile Lys GlnAsp Phe Arg Leu Leu Gly Gln Thr Ser Val Asp Arg Leu Leu Gln Leu Ser GlnGly Gln Ala Val Lys Gly Asn Gln Leu Leu Pro Val Ser Leu Val Lys Arg LysThr Thr Leu Ala Pro Asn Thr Gln Thr Ala Ser Pro Arg Ala Leu Ala Asp SerLeu Met Gln Leu Ala Arg Gln Val Ser Arg Leu Glu Ser Gly Gln

In an alternative embodiment, the inserted lacI transgene is aderivative of the native E.coli lacI gene. In one particular embodiment,the lacI derivative gene is the lacI^(Q) gene having the nucleic acidsequence of SEQ ID NO. 11 (Table 12). The lacI^(Q) variant is identicalto the native E.coli lacI gene except that it has a single pointmutation in the −35 region of the promoter which increases the level oflacI repressor by 10-fold in E.coli . See, for example, M P Calos,Nature 274 (5673): 762-65 (1978). TABLE 12 NUCLEIC ACID SEQUENCE OFLACI^(Q) GENEgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtca SEQ IDNO. 11 attcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcgttttac

In still another embodiment, the lacI derivate gene is the lacI^(Q1)gene having the nucleic acid sequence of SEQ ID NO. 12 (Table 13). ThelacI^(Q1) variant has a rearrangement which substitutes a −35 regionwhose nucleotide sequence exactly matches that of the E.coli−35 regionconsensus sequence, resulting in expression that is 100-fold higher thanthe native promoter in E.coli. See, for example, M P Colas & J H Miller,Mol. & Gen. Genet. 183(3): 559-60(1980). TABLE 13 NUCLEIC ACID SEQUENCEOF LACI^(Q1) GENEagcggcatgcatttacgttgacaccacctttcgcggtatggcatgatagcgcccggaagaga SEQ IDNO. 12 gtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcgttttac

In the present invention, the host cell chromosome can be modified byinsertion of at least one nucleic acid sequence containing at least onecopy of a gene encoding a LacI protein, the gene being capable of use bythe cell to, preferably, constitutively express the encoded LacIprotein, and the polynucleotide containing the gene being other than aPlacI-lacI-lacZYA nucleic acid sequence (i.e. a Plac(−) version of thePlacI-lacI-lacZYA operon) or a PlacI-lacI-lacZ polynucleotide (i.e. astructural lac utilization operon gene-containing portion of such aPlac(−) operon, such as an at least partially truncated version of aPlacI-lacI-lacZYA nucleic acid sequence).

The gene encoding the chosen LacI protein is preferably constitutivelyexpressed. This may be accomplished by use of any promoter that isconstitutively expressed in the selected expression host cell. Forexample, a native E.coli PlacI may be operably attached to the selectedLacI coding sequence, or a different constitutively expressed promotermay be operably attached thereto. In some cases, a regulated promotermay be used, provided that the regulated promoter is maintainedthroughout fermentation in a state wherein the LacI protein iscontinually expressed there from. In a particular embodiment, a lac ortac family promoter is utilized in the present invention, includingPlac, Ptac, Ptrc, PtacII, PlacUV5, 1 pp-PlacUV5, lpp-lac, nprM-lac,T71ac, T5lac, T3lac, and Pmac.

Genomic Insertion Sites

Chromosomal insertion may be performed according to any technique knownin the art. For example, see: D S Toder, “Gene replacement inPseudomonas aeruginosa,” Methods in Enzymology 235:466-74 (1994); and JQuandt & M F Hynes, “Versatile suicide vectors which allow directselection for gene replacement in Gram negative bacteria,” Gene127(1):15-21 (1993). Transposon-type insertion techniques such as areknown in the art, followed by selection, may also be used; see, e.g., IY Goryshin & W S Reznikoff, “Tn5 in vitro transposition,” Journal ofBiological Chemistry 273(13):7367-74 (1998). Alternatively, genetransfection by (non-lytic) phage transduction may also be used forchromosomal insertion; see, e.g., J H Miller, Experiments in MolecularGenetics (1972) (Cold Spring Harbor Lab., N.Y.).

Sites within the bacterial expression host cell chromosome that areuseful places in which to insert the lacI gene(s), or derivativethereof, include any location that is are not required for cell functionunder the fermentation conditions used, for example within any genewhose presence, transcription, or expression is important for thehealthy functioning of the cell under the fermentation conditions used.Illustrative examples of such insertion sites include, but are notlimited to: sucrose import and metabolism genes (e.g., sacB), fructoseimport and catabolism genes (e.g., fructokinase genes,1-phosphofructokinase genes), aromatic carbon source import andutilization genes (e.g., anthranilate operon genes, such as antABCgenes, benzoate operon genes, as benABCD genes), beta-lactamase genes(e.g., ampC, bll1, blc genes, blo genes, blp genes), alkalinephosphatase genes (e.g., phoA), nucleobase or nucleotide biosyntheticgenes (e.g., pyrBCDEF genes), amino acid biosynthetic genes (e.g.,proABC genes), aspartate semi-aldehyde dehydrogenase genes (e.g., asd),3-isopropylmalate dehydrogenase genes (e.g., leuB), and anthranilatesynthase genes (e.g., trpE).

In any embodiment in which the genomic insertion has resulted in or isconcomitant with an auxotrophy, then either the host cell will be grownin media supplying an effective replacement metabolite to the cell toovercome (and avoid) the lethal effect, or a replacement gene will beprovided in the host cell that expresses a biocatalyst effective torestore the corresponding prototrophy, e.g., as a selection marker gene.The gene or genes selected for deletion or inactivation (i.e.“knock-out”) in constructing a metabolic auxotroph can be any geneencoding an enzyme that is operative in a metabolic pathway. The enzymecan be one that is involved in the anabolic biosynthesis of moleculesthat are necessary for cell survival. Alternatively, the enzyme can beone that is involved in the catabolic utilization of molecules that arenecessary for cell survival. Preferably, all operative genes encoding agiven biocatalytic activity are deleted or inactivated in order toensure removal of the targeted enzymatic activity from the host cell inconstructing the auxotrophic host cell. Alternatively, the host cell canexhibit a pre-existing auxotrophy (i.e. native auxotrophy), wherein nofurther genetic modification via deletion or inactivation (knock-out)need be performed.

For example, an amino acid biosynthetic gene (e.g., a proA, proB, orproC gene) or a nucleobase or nucleotide biosynthetic gene (e.g., pyrB,pyrC, pyrD, pyrE, or pyrF) may be used as the insertion site, in whichcase a necessary biosynthetic activity is normally disrupted, thusproducing an auxotrophy. In such a case, either: 1) the medium issupplemented to avoid metabolic reliance on the biosynthetic pathway, aswith a proline or uracil supplement; or 2) the auxotrophic host cell istransformed with a further gene that is expressed and thus replaces thebiocatalyst(s) missing from the biosynthetic pathway, thereby restoringprototrophy to the cell, as with a metabolic selection marker gene suchas proC, pyrF, or thyA. In a particular embodiment, the lacI transgene,or variant thereof, is inserted into a cell that is concomitantly orsubsequently auxotrophically induced through the knock-out of a gene, orcombination of genes, selected from the group consisting of pyrF, thyA,and proC. In a specific embodiment, a native E.coli lacI, lacI^(Q), orlacI^(Q1) transgene is inserted into a cell that is concomitantly orsubsequently rendered auxotrophic through the knock-out of pyrF. Inanother specific embodiment, a native E.coli lacI, lacI^(Q) ,orlacI^(Q1) transgene is inserted into a cell that is concomitantly ofsubsequently rendered auxotrophic through the knock-out of proC. Instill a further embodiment, a native E.coli lacI, lacI^(Q), or lacI^(Q1)transgene is inserted into a cell that is concomitantly or subsequentlyrendered auxotrophic through the knock-out of pyrF and proC.

In another embodiment, a native E.coli lacI, lacI^(Q), or lacI^(Q1)transgene, or derivative thereof, can be inserted into the Levansucraselocus of the host cell. For example, in one particular embodiment, anative E.coli lacI, lacI^(Q), or lacI^(Q1) transgene, or derivativethereof, can be inserted in the Levansucrase gene locus of Pseudomonasfluorescens. In particular, a native E.coli lacI, lacI^(Q), or lacI^(Q1)transgene, or derivative thereof, can be inserted into the Levansucrasegene locus of Pseudomonas fluorescens having the nucleic acid sequenceof SEQ ID. NO. 13 (Table 14). TABLE 14 OPEN READING FRAME OF PF LEVANSUCRASE GENE LOCUSctacccagaacgaagatcagcgcctcaatggcctcaaggttctactggtcgatgattcagcc SEQ IDNO. 13 gaagtcgttgaggtgctgaacatgctgctggaaatggaaggcgcccaagtgagcgccttcagcgaccctttgagcgcgcttgaaacagcccgggatgcccattacgacgtgattatttcggacatcggcatgccgaaaatgaatggccatgagctgatgcagaagctgcgtaaagtaggccaccttcgacaggctcccgccatcgccttaacgggctatggcgctggcaatgaccagaaaaaggcgactgaatcgggctttaatgcgcatgtcagcaaacccgttggccatgattcgctcatcaccttgatcgaaaaactgtgccgctcccgcccctaggcgtggggcaggcgttcaagggtagatgaactgagaaaagcgcacggacgcgcccgtttctggtcgcgacacctgggtatccacgctgcccaccgtgtcgctgcgcaaggtcaggtacaacacggcctggccggcgctgtcactcagcatccagacgctcacaccctccccggccgccctggccttgagcggctgaggctgcagcatctcgatattgaaaccgcgcagcagctcaccgctcaactcgacctccaggggttcctgggccttaccttgcacatgaatcaccagcccatcggaggcgccattgcgcaaaaagcgttggtactccacgcgcaactgcccatcggcactgcgcacctcgcggctgctcagcggcccgctggaaaacagccctgccaagctcaagccgatcagcaccagcagcgcgtaccaacccacccgctcaaagcgccagaccttgcgctgcaaggccatgttttcctgcaccggataattgcggctgtgtaagtcgtcagggtctgggttgttcatagcggggcccggactcaacccttgctgtgctcgggagaagacggccccttggtgacaccccgtgggccggcaatcgcccatatcgcagcgcccagaaacggcagcaccacgactaccgcactccagcctgccttgctggccgaggcgttatcgctgcgccagatgctgttgatgatccacgcatcgagcagtacgaggatcactgccaggcctatccagaagtaagtggtttgcatgatgcacctccaggttatgtaacttttggtgcgcgggtgcgggcagggttcattatttttaggttctctgcctggcgcttggtttgccgccatcatgcgggcaacttcgccgatctacttaatgatcgaacctcttcaaacaagacaagctgaaacgtctcagctcctataaaaagccaaatcatgcacaaatgcattttttgccttgaccacgggaatcgagtcttctaaagtcaaatcactgtatatgaatacagtaatttgattcccttcatggacgagacttactatgaaaagcaccccttcgaaatttggcaaaacaccccatcaacccagcctgtggacccgcgccgatgcgcttaaagtgcatgcggacgaccccaccaccacccagccgctggtcagcgcgaacttcccggtattgagtgacgaggtgtttatctgggacaccatgccgctgcgtgatatcgacggcaacatcacctccgtcgatggctggtcggtgatcttcaccctcaccgcggatcgccacccgaacgacccgcaatacctcgatcagaatggcaactacgacgtcatccgcgactggaacgatcgccatggccgggcaaagatgtactactggttctcccgcaccggcaaagactggaagctcggcggccgagtgatggctgaaggggtttcgcccaccgtgcgcgaatgggccggcacgccgatcctgttgaacgagcaaggcgaagtagacctgtactacaccgccgtcacgcccggcgcgaccatcgtcaaggtgcgtggccgcgtggtgaccaccgagcatggcgtcagcctggtgggctttgagaaggtcaagccgctgttcgaggcggacggcaagatgtaccagaccgaagcgcaaaatgcgttctggggctttcgcgatccatggccgttccgcgacccgaaagacggcaagctgtacatgctgttcgaaggtaacgtggccggcgaacgcggctcgcacaaggtcggtaaagccgaaatcggcgacgtgccgccaggttatgaagacgtcggtaactcgcgcttccagactgcctgcgtcggtatcgccgtggcccgcgacgaagacggcgacgactgggaaatgctgccaccgctgctgaccgcggtgggcgtcaacgaccagaccgaacgcccgcacttcgtgttccaggacggcaagtactacctgttcaccatcagccacaccttcacctacgccgacggcgtgaccggcccggacggcgtgtacggcttcgtcgccgattcgctgttcggtccgtatgtgccgttgaacggctctggtctggtactgggcaacccgtcctcccaaccgttccagacctactcgcactgcgtcatgcccaacggcctggtgacctccttcatcgacagcgtaccgaccgacgacaccggcacgcagatccgtatcggcggcaccgaagcaccgacggtgggcatcaagatcaaagggcagcaaacgtttgtggtcgctgagtatgactacggttacatcccgccgatgctcgacgttacgctcaagtaaccggaggctatgaggtagcggctttgagctcgatgacaaacccgcggtgaatattcgctgcacctgtggcgagggagcttgctcccggttgggccggacagccgccatcgcaggcaagccagctcccacattttggttcctggggcgtcagggaggtatgtgtcggctgaggggccgtcacgggagcaagctccctcgccacaggttcaacagcccattgggtggatattcaggaaatagaaatgcctgcaccattgagttgagtc

IV. LacO Sequences

Attempts to repress the leakiness of a promoter must be balanced by thepotential concomitant reduction in target recombinant polypeptideexpression. One approach to further repress a promoter and reduce theleakiness of the promoters is to modify regulatory elements known asoperator sequences, to increase the capacity of the associated repressorprotein to bind to the operator sequence without reducing the potentialexpression of the target recombinant polypeptide upon induction.

It has been discovered that the use of a dual lac operator inPseudomonas fluorescens offers superior repression of pre-inductionrecombinant protein expression without concomitant reductions in inducedprotein yields.

In one embodiment, a Pseudomonad organism is provided comprising anucleic acid construct containing a nucleic acid comprising at least onelacO sequence involved in the repression of transgene expression. In aparticular embodiment, the Pseudomonad host cell is Pseudomonadfluorescens. In one embodiment, the nucleic acid construct comprisesmore than one lacO sequence. In another embodiment, the nucleic acidconstruct comprises at least one, and preferably more than one, lacOidsequence. In one embodiment, the nucleic acid construct comprises a lacOsequence, or derivative thereof, located 3′ of a promoter, and a lacOsequence, or derivative thereof, located 5′ of a promoter. In aparticular embodiment, the lacO derivative is a lacOid sequence.

In another embodiment of the present invention, nucleic acid constructscomprising more than one lac operator sequence, or derivative thereoffor use in a Pseudomonad host cell is provided. In one embodiment, atleast one lac operator sequence may be a lacO_(id) sequence.

The native E.coli lac operator acts to down regulate expression of thelac operon in the absence of an inducer. To this end, the lac operatoris bound by the LacI repressor protein, inhibiting transcription of theoperon. It has been determined that the LacI protein can bindsimultaneously to two lac operators on the same DNA molecule. See, forexample, Muller et al., (1996) “Repression of lac promoter as a functionof distance, phase, and quality of an auxiliary lac operator,”J.Mol.Biol. 257: 21-29. The repression is mediated by thepromoter-proximal operator O₁ and the two auxiliary operators O₂ and O₃,located 401 base pairs downstream of O₁ within the coding region of thelacZ gene and 92 bp upstream of O₁, respectively (See FIG. 4).Replacement of the native E.coli lac operator sequences with an ideallac operator (O_(id)) results in increased repression of the native lacoperon in E.coli . See Muller et al., (1996) “Repression of lac promoteras a function of distance, phase, and quality of an auxiliary lacoperator,” J.Mol.Biol. 257: 21-29.

The lacO sequence or derivative can be positioned in the E.coli nativeO₁ position with respect to a promoter. Alternatively, the lacO sequenceor derivative can be positioned in the E.coli O₃ position with respectto a promoter. In another embodiment, the lacO sequence or derivativecan be located in the E.coli native O₁ position, the native O₃ position,or both with respect to a promoter. In one embodiment, the nucleic acidconstruct contains at least one lacOid sequence either 5′ to thepromoter sequence or 3′ to the promoter sequence. In a particularembodiment, the nucleic acid construct contains a lacOid sequence 3′ ofa promoter, and at least one lacO sequence, or derivative, 5′ of apromoter. In an alternative embodiment, the nucleic acid constructcontains a lacOid sequence 5′ of a promoter, and at least one lacOsequence, or derivative, 3′ of a promoter. In still another embodiment,the nucleic acid construct contains a lacOid sequence both 5′ and 3′ ofa promoter.

In a particular embodiment, the laco sequence is lacOid represented bySEQ ID NO. 14, or a sequence substantially homologous. In anotherembodiment, a lacOid sequence of SEQ. ID. NO. 59, or a sequencesubstantially homologous to SEQ ID NO. 59 is employed. TABLE 15 LACOIDSEQUENCE 5′-AATTGTGAGCGCTCACAATT-3′ SEQ ID NO. 145′-tgtgtggAATTGTGAGCGCTCACAATTccaca SEQ ID NO. 59 ca-3′V. Isolated Nucleic Acids and Amino Acids

In another aspect of the present invention, nucleic acid sequences areprovided for use in the improved production of proteins.

In one embodiment, nucleic acid sequences encoding prototrophy-restoringenzymes for use in an auxotrophic Pseudomonad host cells are provided.In a particular embodiment, nucleic acid sequences encoding nitrogenousbase compound biosynthesis enzymes purified from the organismPseudomonas fluorescens are provided. In one embodiment, nucleic acidsequences encoding the pyrF gene in Pseudomonas fluorescens is provided(SEQ. ID No.s 1 and 3). In another embodiment, a nucleic acid sequenceencoding the thyA gene in Pseudomonas fluorescens is provided (SEQ. ID.No. 4). In still another embodiment, nucleic acid sequences encoding anamino acid biosynthetic compound purified from the organism Pseudomonasfluorescens are provided. In a particular embodiment, a nucleic acidsequence encoding the proC gene in Pseudomonas fluorescens is provided(SEQ. ID No.s 6 and 8).

In another aspect, the present invention provides novel amino acidsequences for use in the improved production of proteins.

In one embodiment, amino acid sequences of nitrogenous base compoundbiosynthesis enzymes purified from the organism Pseudomonas fluorescensare provided. In one embodiment, the amino acid sequence containing SEQ.ID No. 2 is provided. In another embodiment, an amino acid sequencecontaining SEQ. ID. No. 5 is provided. In still another embodiment,amino acid sequences of an amino acid biosynthetic compound purifiedfrom the organism Pseudomonas fluorescens is provided. In a particularembodiment, an amino acid sequence containing SEQ. ID No. 7 is provided.

One embodiment of the present invention is novel isolated nucleic acidsequences of the Pseudomonas fluorescens pyrF gene (Table 2, Seq. ID No.1; Table 4, Seq. ID No. 3). Another aspect of the present inventionprovides isolated peptide sequences of the Pseudomonas fluorescens pyrFgene (Table 3, Seq. ID No. 2). Nucleic and amino acid sequencescontaining at least 90, 95, 98 or 99% homologous to Seq. ID Nos. 1, 2,or 3 are provided. In addition, nucleotide and peptide sequences thatcontain at least 10, 15, 17, 20 or 25, 30, 40, 50, 75, 100, 150, 250,350, 500, or 1000 contiguous nucleic or amino acids of Seq ID Nos 1, 2,or 3 are also provided. Further provided are fragments, derivatives andanalogs of Seq. ID Nos. 1, 2, or 3. Fragments of Seq. ID Nos. 1, 2, or 3can include any contiguous nucleic acid or peptide sequence thatincludes at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp, 500bp, 1 kbp, 5 kbp or 10 kpb.

Another embodiment of the present invention is novel isolated nucleicacid sequences of the Pseudomonas fluorescens thyA gene (Table 5, Seq.ID No. 4). Another aspect of the present invention provides isolatedpeptide sequences of the Pseudomonas fluorescens thyA gene (Table 6,Seq. ID No. 5). Nucleic and amino acid sequences containing at least 90,95, 98 or 99% homologous to Seq. ID Nos. 4 or 5 are provided. Inaddition, nucleotide and peptide sequences that contain at least 10, 15,17, 20 or 25, 30, 40, 50, 75, 100, 150, 250, 350, 500, or 1000contiguous nucleic or amino acids of Seq ID Nos 4 or 5 are alsoprovided. Further provided are fragments, derivatives and analogs ofSeq. ID Nos. 4 or 5. Fragments of Seq. ID Nos. 4 or 5 can include anycontiguous nucleic acid or peptide sequence that includes at least about10 bp, 15 bp, 17 bp, 20 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 5 kbp or 10

Another embodiment of the present invention is novel isolated nucleicacid sequences of the Pseudomonas fluorescens proC gene (Table 7, Seq.ID No. 6; Table 9, Seq. ID. No. 8). Another aspect of the presentinvention provides isolated peptide sequences of the Pseudomonasfluorescens proC gene (Table 8, Seq. ID No. 7). Nucleic and amino acidsequences containing at least 90, 95, 98 or 99% homologous to Seq. IDNos. 6, 7, or 8 are provided. In addition, nucleotide and peptidesequences that contain at least 10, 15, 17, 20 or 25, 30, 40, 50, 75,100, 150, 250, 350, 500, or 1000 contiguous nucleic or amino acids ofSeq ID Nos 6, 7, or 8 are also provided. Further provided are fragments,derivatives and analogs of Seq. ID Nos. 6, 7, or 8. Fragments of Seq. IDNos. 6, 7, or 8 can include any contiguous nucleic acid or peptidesequence that includes at least about 10 bp, 15 bp, 17 bp, 20 bp, 50 bp,100 bp, 500 bp, 1 kbp, 5 kbp or 10 kpb.

Sequence Homology

Sequence homology is determined according to any of various methods wellknown in the art. Examples of useful sequence alignment and homologydetermination methodologies include those described below.

Alignments and searches for homologous sequences can be performed usingthe U.S. National Center for Biotechnology Information (NCBI) program,MegaBLAST (currently available at http://www.ncbi.nlm.nih.gov/BLAST/).Use of this program with options for percent identity set at 70% foramino acid sequences, or set at 90% for nucleotide sequences, willidentify those sequences with 70%, or 90%, or greater homology to thequery sequence. Other software known in the art is also available foraligning and/or searching for homologous sequences, e.g., sequences atleast 70% or 90% homologous to an information string containing apromoter base sequence or activator-protein-encoding base sequenceaccording to the present invention. For example, sequence alignments forcomparison to identify sequences at least 70% or 90% homologous to aquery sequence can be performed by use of, e.g., the GAP, BESTFIT,BLAST, FASTA, and TFASTA programs available in the GCG Sequence AnalysisSoftware Package (available from the Genetics Computer Group, Universityof Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705), with the default parameters as specified therein, plus aparameter for the extent of homology set at 70% or 90%. Also, forexample, the CLUSTAL program (available in the PC/Gene software packagefrom Intelligenetics, Mountain View, Calif.) may be used.

These and other sequence alignment methods are well known in the art andmay be conducted by manual alignment, by visual inspection, or by manualor automatic application of a sequence alignment algorithm, such as anyof those embodied by the above-described programs. Various usefulalgorithms include, e.g.: the similarity search method described in W.R. Pearson & D. J. Lipman, Proc. Nat'l Acad. Sci. USA 85:2444-48 (Apr1988); the local homology method described in T. F. Smith & M. S.Waterman, in Adv. Appl. Math. 2:482-89 (1981) and in J. Molec. Biol.147:195-97 (1981); the homology alignment method described in S. B.Needleman & C. D. Wunsch, J. Molec. Biol. 48(3):443-53 (Mar 1970); andthe various methods described, e.g., by W. R. Pearson, in Genomics11(3):635-50 (Nov 1991); by W. R. Pearson, in Methods Molec. Biol.24:307-31 and 25:365-89 (1994); and by D. G. Higgins & P. M. Sharp, inComp. Appl'ns in Biosci. 5:151-53 (1989) and in Gene 73(1):237-44 (15Dec 1988).

Nucleic acid hybridization performed under highly stringenthybridization conditions is also a useful technique for obtainingsufficiently homologous sequences for use herein.

VI. Nucleic Acid Constructs

In still another aspect of the present invention, nucleic acidconstructs are provided for use in the improved production of peptides.

In one embodiment, a nucleic acid construct for use in transforming aPseudomonad host cell comprising a) a nucleic acid sequence encoding arecombinant polypeptide, and b) a nucleic acid sequence encoding aprototrophy-enabling enzyme is provided. In another embodiment, thenucleic acid construct further comprises c) a Plac-Ptac family promoter.In still another embodiment, the nucleic acid construct furthercomprises d) at least one lacO sequence, or derivative, 3′ of a lac ortac family promoter. In yet another embodiment, the nucleic acidconstruct further comprises e) at least one lacO sequence, orderivative, 5′ of a lac or tac family promoter. In one embodiment, thederivative lacO sequence can be a lacOid sequence. In a particularembodiment, the Pseudomonad organism is Pseudomonas fluorescens.

In one embodiment of the present invention, nucleic acid constructs areprovided for use as expression vectors in Pseudomonad organismscomprising a) a nucleic acid sequence encoding a recombinantpolypeptide, b) a Plac-Ptac family promoter, c) at least one lacOsequence, or derivative, 3′ of a lac or tac family promoter, d) at leastone lacO sequence, or derivative, 5′ of a lac or tac family promoter. Inone embodiment, the derivative lacO sequence can be a lacOid sequence.In one embodiment, the nucleic acid construct further comprises e) aprototrophy-enabling selection marker for use in an auxotrophicPseudomonad cell. In a particular embodiment, the Pseudomonad organismis Pseudomonas fluorescens.

In one embodiment of the present invention, a nucleic acid construct isprovided comprising nucleic acids that encode at least one biosyntheticenzyme capable of transforming an auxotrophic host cell to prototrophy.The biosynthetic enzyme can be any enzyme capable of allowing anauxotrophic host cell to survive on a selection medium that, without theexpression of the biosynthetic enzyme, the host cell would be incapableof survival due to the auxotrophic metabolic deficiency. As such, thebiosynthetic enzyme can be an enzyme that complements the metabolicdeficiency of the auxotrophic host by restoring prototrophic ability togrow on non-auxotrophic metabolite supplemented media.

In one particular embodiment, the present invention provides a nucleicacid construct that encodes a functional orotodine-5′-phosphatedecarboxylase enzyme that complements an pyrF(−) auxotrophic host. In aparticular embodiment, the nucleic acid construct contains the nucleicacid sequence of SEQ ID NO. 1 or 3. In an alternative embodiment, thenucleic acid construct contains a nucleic acid sequence that encodes theamino acid sequence of SEQ ID NO. 2.

In another particular embodiment, the present invention provides anucleic acid construct that encodes a functional thymidylate synthaseenzyme that complements a thyA (−) auxotrophic host. In a particularembodiment, the nucleic acid construct contains the nucleic acidsequence of SEQ ID NO. 4. In an alternative embodiment, the nucleic acidconstruct contains a nucleic acid sequence that encodes the amino acidsequence of SEQ ID NO. 5.

In a further particular embodiment, the present invention provides anucleic acid construct that encodes a functionalΔ¹-pyrroline-5-carboxylate reductase enzyme that complements a proC (−)auxotrophic host. In a particular embodiment, the nucleic acid constructcontains the nucleic acid sequence of SEQ ID NO. 6 or 8. In analternative embodiment, the nucleic acid construct contains the nucleicacid sequence that encodes the amino acid sequence of SEQ ID NO. 7.

In an alternative embodiment, the present invention provides a nucleicacid construct that encodes at least one biosynthetic enzyme capable oftransforming an auxotrophic host cell to prototrophy and an additionalnon-auxotrophic selection marker. Examples of non-auxotrophic selectionmarkers are well known in the art, and can include markers that giverise to colorimetric/chromogenic or a luminescent reaction such as lacZgene, the GUS gene, the CAT gene, the luxAB gene, antibiotic resistanceselection markers such as amphotericin B, bacitracin, carbapenem,cephalosporin, ethambutol, fluoroquinolones, isonizid, cephalosporin,methicillin, oxacillin, vanomycin, streptomycin, quinolines, rifampin,rifampicin, sulfonamides, ampicillin, tetracycline, neomycin,cephalothin, erythromycin, streptomycin, kanamycin, gentamycin,penicillin, and chloramphenicol resistance genes, or other commonly usednon-auxotrophic selection markers.

In another embodiment, the expression vector can comprise more than onebiosynthetic enzyme capable of transforming an auxotrophic host cell toprototrophy. The biosynthetic enzymes can be any enzymes capable ofallowing an auxotrophic host cell to survive on a selection medium that,without the expression of the biosynthetic enzyme, the host cell wouldbe incapable of survival due to the auxotrophic metabolic deficiency. Assuch, the biosynthetic enzymes can be enzymes that complement themetabolic deficiencies of the auxotrophic host by restoring prototrophicability to grow on non-auxotrophic metabolite supplemented media. Forexample, an expression vector comprise a first and secondprototrophy-enabling selection marker gene, allowing the host cellcontaining the construct to be maintained under either or both of theconditions in which host cell survival requires the presence of theselection marker gene(s). When only one of the marker-gene dependentsurvival conditions is present, the corresponding marker gene must beexpressed, and the other marker gene(s) may then be either active orinactive, though all necessary nutrients for which the cell remainsauxotrophic will still be supplied by the medium. This permits the sametarget gene, or the same set of covalently linked target genes, encodingthe desired transgenic product(s) and/or desired transgenicactivity(ies), to be maintained in the host cell continuously as thehost cell is transitioned between or among different conditions. Thecoding sequence of each of the chosen selection marker genesindependently can be operatively attached to either a constitutive or aregulated promoter.

In a particular embodiment, the nucleic acid vector comprises a nucleicacid construct that encodes a functional orotodine-5′-phosphatedecarboxylase enzyme and a functional Δ¹-pyrroline-5-carboxylatereductase enzyme that can complement a pyrF(−) auxotrophic host cell, aproC(−) auxotrophic host cell, or a pyrF(−)/proC(−) dual-auxotrophichost cell. In a particular embodiment, the nucleic acid constructcomprises the nucleic acid sequences of SEQ ID NO. 1 or 3, and SEQ ID.NO. 6 or 8. In an alternative embodiment, the nucleic acid constructcontains a nucleic acid sequence that encodes the amino acid sequencesof SEQ ID NO. 2 and 7.

In an alternative particular embodiment, the nucleic acid vectorcomprises a nucleic acid construct that encodes a functionalorotodine-5′-phosphate decarboxylase enzyme and a functional thymidylatesynthase enzyme that can complement a pyrF(−) auxotrophic host cell, athyA(−) auxotrophic host cell, or a pyrF(−)/thyA(−) dual-auxotrophichost cell. In a particular embodiment, the nucleic acid constructcomprises the nucleic acid sequences of SEQ ID NO. 1 or 3, and SEQ ID.NO. 4. In an alternative embodiment, the nucleic acid construct containsa nucleic acid sequence that encodes the amino acid sequences of SEQ IDNO. 2 and 5.

In a particular embodiment, the nucleic acid vector comprises a nucleicacid construct that encodes a functional Δ¹-pyrroline-5-carboxylatereductase enzyme and a thymidylate synthase enzyme that can complement aproC(−) auxotrophic host cell, a thyA(−) auxotrophic host cell, or aproC(−)/thyA(−) dual-auxotrophic host cell. In a particular embodiment,the nucleic acid construct comprises the nucleic acid sequences of SEQID NO. 4, and SEQ ID. NO. 6 or 8. In an alternative embodiment, thenucleic acid construct contains a nucleic acid sequence that encodes theamino acid sequences of SEQ ID NO. 5 and 7.

Promoters

In a fermentation process, once expression of the target recombinantpolypeptide is induced, it is ideal to have a high level of productionin order to maximize efficiency of the expression system. The promoterinitiates transcription and is generally positioned 10-100 nucleotidesupstream of the ribosome binding site. Ideally, a promoter will bestrong enough to allow for recombinant polypeptide accumulation ofaround 50% of the total cellular protein of the host cell, subject totight regulation, and easily (and inexpensively) induced.

The promoters used in accordance with the present invention may beconstitutive promoters or regulated promoters. Examples of commonly usedinducible promoters and their subsequent inducers include lac (IPTG),lacUV5 (IPTG), tac (IPTG), trc (IPTG), P_(syn) (IPTG), trp (tryptophanstarvation), araBAD (1-arabinose), 1pp^(a) (IPTG), 1pp-lac (IPTG), phoA(phosphate starvation), recA (nalidixic acid), proU (osmolarity), cst-1(glucose starvation), teta (tretracylin), cada (pH), nar (anaerobicconditions), PL (thermal shift to 42° C.), cspA (thermal shift to 20°C.), T7 (thermal induction), T7-lac operator (IPTG), T3-lac operator(IPTG), T5-lac operator (IPTG), T4 gene32 (T4 infection), nprM-lacoperator (IPTG), Pm (alkyl- or halo-benzoates), Pu (alkyl- orhalo-toluenes), Psa1 (salicylates), and VHb (oxygen). See, for example,Makrides, S. C. (1996) Microbiol. Rev. 60, 512-538; Hannig G. &Makrides, S. C. (1998) TIBTECH 16, 54-60; Stevens, R. C. (2000)Structures 8, R177-R185. See, e.g.: J. Sanchez-Romero & V. De Lorenzo,Genetic Engineering of Nonpathogenic Pseudomonas strains as Biocatalystsfor Industrial and Environmental Processes, in Manual of IndustrialMicrobiology and Biotechnology (A. Demain & J. Davies, eds.) pp.460-74(1999) (ASM Press, Washington, D.C.); H. Schweizer, Vectors to expressforeign genes and techniques to monitor gene expression forPseudomonads, Current-Opinion in Biotechnology, 12:439-445 (2001); andR. Slater & R. Williams, The Expression of Foreign DNA in Bacteria, inMolecular Biology and Biotechnology (J. Walker & R. Rapley, eds.)pp.125-54 (2000) (The Royal Society of Chemistry, Cambridge, UK).

A promoter having the nucleotide sequence of a promoter native to theselected bacterial host cell can also be used to control expression ofthe transgene encoding the target polypeptide, e.g, a Pseudomonasanthranilate or benzoate operon promoter (Pant, Pben). Tandem promotersmay also be used in which more than one promoter is covalently attachedto another, whether the same or different in sequence, e.g., a Pant-Pbentandem promoter (interpromoter hybrid) or a Plac-Plac tandem promoter.

Regulated promoters utilize promoter regulatory proteins in order tocontrol transcription of the gene of which the promoter is a part. Wherea regulated promoter is used herein, a corresponding promoter regulatoryprotein will also be part of an expression system according to thepresent invention. Examples of promoter regulatory proteins include:activator proteins, e.g., E.coli catabolite activator protein, MalTprotein; AraC family transcriptional activators ; repressor proteins,e.g., E.coli LacI proteins; and dual-faction regulatory proteins, e.g.,E.coli NagC protein. Many regulated-promoter/promoter-regulatory-proteinpairs are known in the art.

Promoter regulatory proteins interact with an effector compound, i.e. acompound that reversibly or irreversibly associates with the regulatoryprotein so as to enable the protein to either release or bind to atleast one DNA transcription regulatory region of the gene that is underthe control of the promoter, thereby permitting or blocking the actionof a transcriptase enzyme in initiating transcription of the gene.Effector compounds are classified as either inducers or co-repressors,and these compounds include native effector compounds and gratuitousinducer compounds. Manyregulated-promoter/promoter-regulatory-protein/effector-compound triosare known in the art. Although an effector compound can be usedthroughout the cell culture or fermentation, in a particular embodimentin which a regulated promoter is used, after growth of a desiredquantity or density of host cell biomass, an appropriate effectorcompound is added to the culture in order to directly or indirectlyresult in expression of the desired target gene(s).

By way of example, where a lac family promoter is utilized, a lacI gene,or derivative thereof such as a lacI^(Q) or lacI^(Q1) gene, can also bepresent in the system. The lacI gene, which is (normally) aconstitutively expressed gene, encodes the Lac repressor protein (LacIprotein) which binds to the lac operator of these promoters. Thus, wherea lac family promoter is utilized, the lacI gene can also be includedand expressed in the expression system. In the case of the lac promoterfamily members, e.g., the tac promoter, the effector compound is aninducer, preferably a gratuitous inducer such as IPTG(isopropyl-β-D-1-thiogalactopyranoside, also called“isopropylthiogalactoside”).

In a particular embodiment, a lac or tac family promoter is utilized inthe present invention, including Plac, Ptac, Ptrc, PtacII, PlacUV5,Ipp-PlacUV5, Ipp-lac, nprM-lac, T71ac, T51ac, T31ac, and Pmac.

Other Elements

Other regulatory elements can be included in an expression construct,including lacO sequences and derivatives, as discussed above. Suchelements include, but are not limited to, for example, transcriptionalenhancer sequences, translational enhancer sequences, other promoters,activators, translational start and stop signals, transcriptionterminators, cistronic regulators, polycistronic regulators, tagsequences, such as nucleotide sequence “tags” and “tag” peptide codingsequences, which facilitates identification, separation, purification,or isolation of an expressed polypeptide, including His-tag, Flag-tag,T7-tag, S-tag, HSV-tag, B-tag, Strep-tag, polyarginine, polycysteine,polyphenylalanine, polyaspartic acid, (Ala-Trp-Trp-Pro)n, thioredoxin,beta-galactosidase, chloramphenicol acetyltransferase, cyclomaltodextringluconotransferase, CTP:CMP-3-deoxy-D-manno-octulosonatecytidyltransferase, trpE or trpLE, avidin, streptavidin, T7 gene 10, T4gp55, Staphylococcal protein A, streptococcal protein G, GST, DHFR, CBP,MBP, galactose binding domain, Calmodulin binding domain, GFP, KSI,c-myc, ompT, ompA, pelB, , NusA, ubiquitin, and hemosylin A.

At a minimum, a protein-encoding gene according to the present inventioncan include, in addition to the protein coding sequence, the followingregulatory elements operably linked thereto: a promoter, a ribosomebinding site (RBS), a transcription terminator, translational start andstop signals. Useful RBSs can be obtained from any of the species usefulas host cells in expression systems according to the present invention,preferably from the selected host cell. Many specific and a variety ofconsensus RBSs are known, e.g., those described in and referenced by D.Frishman et al., Starts of bacterial genes: estimating the reliabilityof computer predictions, Gene 234(2):257-65 (8 Jul. 1999); and B. E.Suzek et al., A probabilistic method for identifying start codons inbacterial genomes, Bioinformatics 17(12):1123-30 (December 2001). Inaddition, either native or synthetic RBSs may be used, e.g., thosedescribed in: EP 0207459 (synthetic RBSs); 0. Ikehata et al., Primarystructure of nitrile hydratase deduced from the nucleotide sequence of aRhodococcus species and its expression in Escherichia coli, Eur. J.Biochem. 181(3):563-70 (1989) (native RBS sequence of AAGGAAG). Furtherexamples of methods, vectors, and translation and transcriptionelements, and other elements useful in the present invention aredescribed in, e.g.: U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat. No.5,128,130 to Gilroy et al.; U.S. Pat. No. 5,281,532 to Rammler et al.;U.S. Pat. Nos. 4,695,455 and 4,861,595 to Barnes et al.; U.S. Pat.No.4,755,465 to Gray et al.; and U.S. Pat. No. 5,169,760 to Wilcox.

Vectors

Transcription of the DNA encoding the enzymes of the present inventionby a Pseudomonad host can further be increased by inserting an enhancersequence into the vector or plasmid. Typical enhancers are cis-actingelements of DNA, usually about from 10 to 300 bp in size that act on thepromoter to increase its transcription.

Generally, the recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of thePseudomonad host cell, e.g., the prototrophy restoring genes of thepresent invention, and a promoter derived from a highly-expressed geneto direct transcription of a downstream structural sequence. Suchpromoters have been described above. The heterologous structuralsequence is assembled in appropriate phase with translation initiationand termination sequences, and in certain embodiments, a leader sequencecapable of directing secretion of the translated polypeptide.Optionally, and in accordance with the present invention, theheterologous sequence can encode a fusion polypeptide including anN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct.

Useful expression vectors for use with P. fluorescens in expressingenzymes are constructed by inserting a structural DNA sequence encodinga desired target polypeptide together with suitable translationinitiation and termination signals in operable reading phase with afunctional promoter. The vector will comprise one or more phenotypicselectable markers and an origin of replication to ensure maintenance ofthe vector and to, if desirable, provide amplification within the host.Suitable hosts for transformation in accordance with the presentdisclosure include various species within the genera Pseudomonas, andparticularly particular is the host cell strain of Pseudomonasfluorescens.

Vectors are known in the art as useful for expressing recombinantproteins in host cells, and any of these may be modified and used forexpressing the genes according to the present invention. Such vectorsinclude, e.g., plasmids, cosmids, and phage expression vectors. Examplesof useful plasmid vectors that can be modified for use on the presentinvention include, but are not limited to, the expression plasmidspBBR1MCS, pDSK519, pKT240, pML122, pPS10 , RK2, RK6, pRO1600, andRSF1010. Further examples can include pALTER-Ex1, pALTER-Ex2, pBAD/His,pBAD/Myc-His, pBAD/gIII, pCal-n, pCal-n-EK, pCal-c, pCal-Kc, pcDNA 2.1,pDUAL, pET-3a-c, pET 9a-d, pET-11a-d, pET-12a-c, pET-14b, pET15b,pET-16b, pET-17b, pET-19b, pET-20b(+), pET-21a-d(+), pET-22b(+),pET-23a-d(+), pET24a-d(+), pET-25b(+), pET-26b(+), pET-27b(+),pET28a-c(+), pET-29a-c(+), pET-30a-c(+), pET31b(+), pET-32a-c(+),pET-33b(+), pET-34b(+), pET35b(+), pET-36b(+), pET-37b(+), pET-38b(+),pET-39b(+), pET-40b(+), pET41la-c(+), pET-42a-c(+pET43a-c(+), pETBlue-1,pETBlue-2, pETBlue-3, pGEMEX-1, pGEMEX-2, pGEX1λT, pGEX-2T, pGEX-2TK,pGEX-3X, pGEX4T, pGEX-5X, pGEX-6P, pHAT10/11/12, pHAT20, pHAT-GFPuv,pKK223-3, pLEX, pMAL-c2X, pMAL-c2E, pMAL-c2g, pMAL-p2X, pMAL-p2E,pMAL-p2G, pProEX HT, pPROLar.A, pPROTet.E, pQE-9, pQE-16, pQE-30/31/32,pQE40, pQE-50, pQE-70, pQE-80/81/82L, pQE-100, pRSET, and pSE280,pSE380, pSE420, pThioHis, pTrc99A, pTrcHis, pTrcHis2, pTriEx-1,pTriEx-2, pTrxFus. Other examples of such useful vectors include thosedescribed by, e.g.: N. Hayase, in Appl. Envir. Microbiol. 60(9):3336-42(September 1994); A. A. Lushnikov et al., in Basic Life Sci. 30:657-62(1985); S. Graupner & W. Wackernagel, in Biomolec. Eng. 17(1):11-16.(October 2000); H. P. Schweizer, in Curr. Opin. Biotech. 12(5):439-45(October 2001); M. Bagdasarian &. K. N. Timmis, in Curr. TopicsMicrobiol. Immunol. 96:47-67 (1982); T. Ishii et al., in FEMS Microbiol.Lett. 116(3):307-13 (Mar 1, 1994); I. N. Olekhnovich & Y. K. Fomichev,in Gene 140(1):63-65 (Mar 11, 1994); M. Tsuda & T. Nakazawa, in Gene136(1-2):257-62 (Dec. 22, 1993); C. Nieto et al., in Gene 87(1):145-49(Mar 1, 1990); J. D. Jones & N. Gutterson, in Gene 61(3):299-306 (1987);M. Bagdasarian et al., in Gene 16(1-3):237-47 (December 1981); H. P.Schweizer et al., in Genet. Eng. (NY) 23:69-81 (2001); P. Mukhopadhyayet al., in J. Bact. 172(1):477-80 (January 1990); D. O. Wood et al., inJ. Bact. 145(3):1448-51 (March 1981); Holtwick et al., in Microbiology147(Pt 2):337-44 (Febuary 2001).

Further examples of expression vectors that can be useful in Pseudomonashost cells include those listed in Table 16 as derived from theindicated replicons. TABLE 16 SOME EXAMPLES OF USEFUL EXPRESSION VECTORSReplicon Vector(s) _(P)PS10 _(P)CN39, _(P)CN51 RSF1010 _(P)KT261-3_(P)MMB66EH _(P)EB8 _(P)PLGN1 _(P)MYC1050 RK2/RP1 _(P)RK415 _(P)JB653_(P)RO1600 _(P)UCP _(P)BSP

The expression plasmid, RSF1010, is described, e.g., by F. Heffron etal., in Proc. Nat'l Acad. Sci. USA 72(9):3623-27 (September 1975), andby K. Nagahari & K. Sakaguchi, in J. Bact. 133(3):1527-29 (March 1978).Plasmid RSF1010O and derivatives thereof are particularly useful vectorsin the present invention. Exemplary, useful derivatives of RSF1010,which are known in the art, include, e.g., pKT212, pKT214, pKT231 andrelated plasmids, and pMYC1050 and related plasmids (see, e.g., U.S.Pat, Nos. 5,527,883 and 5,840,554 to Thompson et al.), such as, e.g.,pMYC1803. Plasmid pMYC1803 is derived from the RSF1010-based plasmidpTJS260 (see U.S. Pat. No. 5,169,760 to Wilcox), which carries aregulated tetracycline resistance marker and the replication andmobilization loci from the RSF1010 plasmid. Other exemplary usefulvectors include those described in U.S. Pat. No. 4,680,264 to Puhler etal.

In a one embodiment, an expression plasmid is used as the expressionvector. In another embodiment, RSF1010 or a derivative thereof is usedas the expression vector. In still another embodiment, pMYC1050 or aderivative thereof, or pMYC1803 or a derivative thereof, is used as theexpression vector.

VII. Expression of Recombinant Polypeptides in an Pseudomonad Host Cells

In one aspect of the present invention, processes of expressingrecombinant polypeptides for use in improved protein production areprovided.

In one embodiment, the process provides expression of a nucleic acidconstruct comprising nucleic acids encoding a) a recombinantpolypeptide, and b) a prototrophy-restoring enzyme in a Pseudomonad thatis auxotrophic for at least one metabolite. In an alternativeembodiment, the Pseudomonad is auxotrophic for more than one metabolite.In one embodiment, the Pseudomonad is a Pseudomonas fluorescens cell. Ina particular embodiment, a recombinant polypeptide is expressed in aPseudomonad that is auxotrophic for a metabolite, or combination ofmetabolites, selected from the group consisting of a nitrogenous basecompound and an amino acid. In a more particular embodiment, recombinantpolypeptides are expressed in a Pseudomonad that is auxotrophic for ametabolite selected from the group consisting of uracil, proline, andthymidine. In another embodiment, the auxotrophy can be generated by theknock-out of the host pyrF, proC, or thyA gene, respectively. Analternative embodiment, recombinant polypeptides are expressed in anauxotrophic Pseudomonad cell that has been genetically modified throughthe insertion of a native E.coli lacI gene, lacI^(Q) gene, or lacI^(Q1)gene, other than as part of the PlacI-lacI-lacZYA operon, into the hostcell's chromosome. In one particular embodiment, the vector containingthe recombinant polypeptide expressed in the auxotrophic host cellcomprises at least two lac operator sequences, or derivatives thereof.In still a further embodiment, the recombinant polypeptide is driven bya Plac family promoter.

In another embodiment, the process involves the use of Pseudomonad hostcells that have been genetically modified to provide at least one copyof a LacI encoding gene inserted into the Pseudomonad host cell'sgenome, wherein the lacI encoding gene is other than as part of thePlacI-lacI-lacZYA operon. In one embodiment, the gene encoding the Lacrepressor protein is identical to that of native E.coli lacI gene. Inanother embodiment, the gene encoding the Lac repressor protein is thelacI^(Q) gene. In still another embodiment, the gene encoding the Lacrepressor protein is the lacI^(Q1) gene. In a particular embodiment, thePseudomonad host cell is Pseudomonas fluorescens. In another embodiment,the Pseudomonad is further genetically modified to produce anauxotrophic cell. In another embodiment, the process producesrecombinant polypeptide levels of at least about 3 g/L, 4 g/L, 5 g/L 6g/L, 7 g/L, 8 g/L, 9 g/L or at least about 10 g/L. In anotherembodiment, the recombinant polypeptide is expressed in levels ofbetween 3 g/L and 100 g/L.

The method generally includes:

-   -   a) providing a Pseudomonad host cell, preferably a Pseudomonas        fluorescens, as described in the present invention,    -   b) transfecting the host cell with at least one nucleic acid        expression vector comprising i) a target recombinant polypeptide        of interest, and, in the case of the utilization of an        auxotrophic host, ii) a gene encoding a prototrophy enabling        enzyme that, when expressed, overcomes the auxotrophy of the        host cell;    -   c) growing the host cell in a growth medium that provides a        selection pressure effective for maintaining the nucleic acid        expression vector containing the recombinant polypeptide of        interest in the host cell; and    -   d) expressing the target recombinant polypeptide of interest.

The method can further comprise transfecting the host cell with at leastonce nucleic acid expression vector further comprising iii) a Placfamily promoter, and optionally iv) more than one lac operatorsequences. In one embodiment, at least one lac operator sequence may bea lac_(O)id sequence. Preferably, the expression system is capable ofexpressing the target polypeptide at a total productivity of polypeptideof at least 1 g/L to at least 80 g/L. In a particular embodiment, therecombinant polypeptide is expressed at a level of at least 3 g/L, 4g/L,5g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 12 g/L, 15 g/L, 20 g/L, 25 gL,30 g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L, or at least 80g/L. In a particular embodiment, a lac or tac family promoter isutilized in the present invention, including Plac, Ptac, Ptrc, PtacII,PlacUV5, 1pp-PlacUV5, 1pp-lac, nprM-lac, T71ac, T51ac, T31ac, and Pmac.

In one embodiment, at least one recombinant polypeptide can be expressedin a Pseudomonad cell that is auxotrophic for one metabolite, whereinthe auxotrophy serves as a selection marker for the maintenance of thenucleic acid expression vector encoding the polypeptide of interest andthe prototrophy-enabling enzyme. Alternatively, more than onerecombinant polypeptide can be expressed in a Pseudomonad cell that isauxotrophic for one metabolite, wherein the nucleic acids encoding therecombinant polypeptides can be contained on the same vector, oralternatively, on multiple vectors.

In yet another embodiment, more than one expression vector encodingdifferent target polypeptides can be maintained in a Pseudomonad hostcell auxotrophic for at least one metabolite, wherein one expressionvector contains a nucleic acid encoding a prototrophic-enabling enzymeand a first target polypeptide of interest, and a second expressionvector contains a nucleic acid encoding an alternative, non-auxotrophicselection marker and a second polypeptide of interest.

In another embodiment, at least one recombinant polypeptide can beexpressed in a Pseudomonad cell that is auxotrophic for more than onemetabolite, wherein the multiple auxotrophies serve as selection markersfor the maintenance of nucleic acid expression vectors. For example, anexpression vector may be utilized in which a first and secondprototrophy-enabling selection marker gene are present. If both markergenes are located on the same DNA construct, the host cell containingthe construct may be maintained under either or both of the conditionsin which host cell survival requires the presence of the selectionmarker gene(s). When only one of the marker-gene dependent survivalconditions is present, the corresponding marker gene must be expressed,and the other marker gene(s) can then be either active or inactive,though all necessary nutrients for which the cell remains auxotrophicwill still be supplied by the medium. This permits the same target gene,or the same set of covalently linked target genes, encoding the desiredtransgenic product(s) and/or desired transgenic activity(ies), to bemaintained in the host cell continuously as the host cell istransitioned between or among different conditions. If each of the twoselection marker genes is located on a different DNA construct, then, inorder to maintain both of the DNA constructs in the host cell, both ofthe marker-gene dependent survival conditions are present, and both ofthe corresponding marker gene must be expressed. This permits more thanone non-covalently linked target gene or set of target gene(s) to beseparately maintained in the host cell. The coding sequence of each ofthe chosen selection marker genes independently can be operativelyattached to either a constitutive or a regulated promoter.

Dual-target-gene examples of such a multi-target-gene system include,but are not limited to: (1) systems in which the expression product ofone of the target genes interacts with the other target gene itself; (2)systems in which the expression product of one of the target genesinteracts with the other target gene's expression product, e.g., aprotein and its binding protein or the α- and β- polypeptides of anαn-βn protein; (3) systems in which the two expression products of thetwo genes both interact with a third component, e.g., a third componentpresent in the host cell; (4) systems in which the two expressionproducts of the two genes both participate in a common biocatalyticpathway; and (5) systems in which the two expression products of the twogenes function independently of one another, e.g., a bi-clonal antibodyexpression system.

In one example of a dual-target-gene system of the above-listed type(1), a first target gene can encode a desired target protein, whereinthe first target gene is under the control of a regulated promoter; thesecond target gene may then encode a protein involved in regulating thepromoter of the first target gene, e.g., the second target gene mayencode the first target gene's promoter activator or repressor protein.In an example in which the second gene encodes a promoter regulatoryprotein for the first gene, the coding sequence of the second gene canbe under the control of a constitutive promoter. In one embodiment, thesecond gene will be part of a separate DNA construct that is amaintained in the cell as a high-copy-number construct with a copynumber of at least 10, 20, 30, 40, 50, or more than 50 copies beingmaintained in the host cell.

In another embodiment, the present invention provides the use of morethan one lacO sequence on an expression vector in the production ofrecombinant polypeptides in Pseudomonads, particularly in Pseudomonasfluorescens

In another aspect, the present invention provides a method of producinga recombinant polypeptide comprising transforming a bacterial host cellthat is a member of the Pseudomonads and closely related bacteria havingat least one chromosomally inserted copy of a Lac repressor proteinencoding a lacI transgene, or derivative thereof such as lacI^(Q1) orlacI^(Q1,) which transgene is other than part of a whole or truncatedstructural gene containing PlacI-lacI-lacZYA construct with a nucleicacid construct encoding at least one target recombinant polypeptide. Thenucleic acid encoding at least one target recombinant polypeptide can beoperably linked to a Plac family promoter, in which all of the Placfamily promoters present in the host cell are regulated by Lac repressorproteins expressed solely from the lacI transgene inserted in thechromosome. Optionally, the expression system is capable of expressingthe target polypeptide at a total productivity of at least 3 g/L to atleast 10 g/L. Preferably, the expression system is capable of expressingthe target polypeptide at a total productivity of polypeptide of atleast 3 g/L, 4g/L, 5g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, or at least 10 g/L.

In one embodiment, the present invention provides a method of expressingrecombinant polypeptides in an expression system utilizing auxotrophicPseudomonads or related bacteria that have been further geneticallymodified to provide at least one copy of a LacI encoding gene insertedinto the cell's genome, other than as part of the PlacI-lacI-lacZYAoperon. In a particular embodiment, a recombinant polypeptide isexpressed in an auxotrophic Pseudomonas fluorescens host cell containinga lacI transgene insert. In another particular embodiment, a recombinantpolypeptide is expressed in an auxotrophic Pseudomonas fluorescens hostcell containing a lacI^(Q1) transgene insert. In still anotherparticular embodiment, a recombinant polypeptide is expressed in anauxotrophic Pseudomonas fluorescens host cell containing a lacI^(Q1)transgene insert. The Pseudomonas fluorescens host can be auxotrophicfor a biochemical required by the cell for survival. In a particularembodiment, the Pseudomonas fluorescens cell is auxotrophic for anitrogenous base. In a particular embodiment, the Pseudomonasfluorescens is auxotrophic for a nitrogenous base selected from thegroup consisting of thymine and uracil. In a particularly particularembodiment, the Pseudomonas fluorescens host cell's auxotrophy isinduced by a genetic modification to a pyrF or thyA gene rendering theassociated encoded product non-functional. In an alternative embodiment,the Pseudomonas fluorescens cell is auxotrophic for an amino acid. In aparticular embodiment, the Pseudomonas fluorescens is auxotrophic forthe amino acid proline. In a particularly particular embodiment, thePseudomonas fluorescens host cell's auxotrophy is induced by a geneticmodification to a proC gene rendering the associated encoded productnon-functional.

Transformation

Transformation of the Pseudomonad host cells with the vector(s) may beperformed using any transformation methodology known in the art, and thebacterial host cells may be transformed as intact cells or asprotoplasts (i.e. including cytoplasts). Exemplary transformationmethodologies include poration methodologies, e.g., electroporation,protoplast fusion, bacterial conjugation, and divalent cation treatment,e.g., calcium chloride treatment or CaCl/Mg²+ treatment, or other wellknown methods in the art. See, e.g., Morrison, J. Bact., 132:349-351(1977); Clark-Curtiss & Curtiss, Methods in Enzymology, 101:347-362 (Wuet al., eds, 1983), Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

Selection

Preferably, cells that are not successfully transformed are selectedagainst following transformation, and continuously during thefermentation. The selection marker can be an auxotrophic selectionmarker or a traditional antibiotic selection marker. When the cell isauxotrophic for multiple nutrient compounds, the auxotrophic cell can begrown on medium supplemented with all of those nutrient compounds untiltransformed with the prototrophy-restoring vector. Where the host cellis or has been made defective for multiple biosynthetic activities, theprototrophy-restorative marker system(s) can be selected to restore oneor more or all of the biosynthetic activities, with the remainder beingcompensated for by continuing to provide, in the medium, thestill-lacking nutrients. In selection marker systems in which more thanone biosynthetic activity, and/or more than one prototrophy, isrestored, the plurality of selection marker genes may be expressedtogether on one vector or may be co-expressed separately on differentvectors. Even where a single metabolite is the target of the selectionmarker system, multiple biosynthetic activities may be involved in theselection marker system. For example, two or more genes encodingactivities from the same anabolic pathway may be expressed together onone vector or may be co-expressed separately on different vectors, inorder to restore prototrophy in regard to biosynthesis of the compoundthat is the product of the pathway.

Where the selection marker is an antibiotic resistance gene, theassociated antibiotic can be added to the medium to select against nontransformed and revertant cells, as well known in the art.

Fermentation

As used herein, the term “fermentation” includes both embodiments inwhich literal fermentation is employed and embodiments in which other,non-fermentative culture modes are employed. Fermentation may beperformed at any scale. In one embodiment, the fermentation medium maybe selected from among rich media, minimal media, a mineral salts media;a rich medium may be used, but is preferably avoided. In anotherembodiment either a minimal medium or a mineral salts medium isselected. In still another embodiment, a minimal medium is selected. Inyet another embodiment, a mineral salts medium is selected. Mineralsalts media are particularly particular.

Prior to transformation of the host cell with a nucleic acid constructencoding a prototrophic enabling enzyme, the host cell can be maintainedin a media comprising a supplemental metabolite, or analogue thereof,that complements the auxotrophy. Following transformation, the host cellcan be grown in a media that is lacking the complementary metabolitethat the host cell is auxotrophic for. In this way, host cells that donot contain the selection marker enabling prototrophy are selectedagainst. Likewise cells expressing recombinant proteins from expressionvectors containing an antibiotic resistance selection marker gene can bemaintained prior to transformation on a medium lacking the associatedantibiotic used for selection. After transformation and during thefermentation, an antibiotic can be added to the medium, atconcentrations known in the art, to select against non-transformed andrevertant cells.

Mineral salts media consists of mineral salts and a carbon source suchas, e.g., glucose, sucrose, or glycerol. Examples of mineral salts mediainclude, e.g., M9 medium, Pseudomonas medium (ATCC 179), Davis andMingioli medium (see, B D Davis & E S Mingioli, in J. Bact. 60:17-28(1950)). The mineral salts used to make mineral salts media includethose selected from among, e.g., potassium phosphates, ammonium sulfateor chloride, magnesium sulfate or chloride, and trace minerals such ascalcium chloride, borate, and sulfates of iron, copper, manganese, andzinc. No organic nitrogen source, such as peptone, tryptone, aminoacids, or a yeast extract, is included in a mineral salts medium.Instead, an inorganic nitrogen source is used and this may be selectedfrom among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia.A particular mineral salts medium will contain glucose as the carbonsource. In comparison to mineral salts media, minimal media can alsocontain mineral salts and a carbon source, but can be supplemented with,e.g., low levels of amino acids, vitamins, peptones, or otheringredients, though these are added at very minimal levels.

In one embodiment, media can be prepared using the components listed inTable 16 below. The components can be added in the following order:first (NH₄)HPO₄, KH₂PO₄ and citric acid can be dissolved inapproximately 30 liters of distilled water; then a solution of traceelements can be added, followed by the addition of an antifoam agent,such as Ucolub N 115. Then, after heat sterilization (such as atapproximately 121° C.), sterile solutions of glucose MgSO₄ andthiamine-HCL can be added. Control of pH at approximately 6.8 can beachieved using aqueous ammonia. Sterile distilled water can then beadded to adjust the initial volume to 371 minus the glycerol stock (123mL). The chemicals are commercially available from various suppliers,such as Merck. This media can allow for high cell density cultivation(HCDC) for growth of Pseudomonas species and related bacteria. The HCDCcan start as a batch process which is followed by two-phase fed-batchcultivation. After unlimited growth in the batch part, growth can becontrolled at a reduced specific growth rate over a period of 3 doublingtimes in which the biomass concentration can increased several fold.Further details of such cultivation procedures is described byRiesenberg, D.; Schulz, V.; Knorre, W. A.; Pohl, H. D.; Korz, D.;Sanders, E. A.; Ross, A.; Deckwer, W. D. (1991) “High cell densitycultivation of Escherichia coli at controlled specific growth rate” JBiotechnol: 20(1) 17-27.

The expression system according to the present invention can be culturedin any fermentation format. For example, batch, fed-batch,semi-continuous, and continuous fermentation modes may be employedherein.

The expression systems according to the present invention are useful fortransgene expression at any scale (i.e. volume) of fermentation. Thus,e.g., microliter-scale, centiliter scale, and deciliter scalefermentation volumes may be used; and 1 Liter scale and largerfermentation volumes can be used. In one embodiment, the fermentationvolume will be at or above 1 Liter. In another embodiment, thefermentation volume will be at or above 5 Liters, 10 Liters, 15 Liters,20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 50Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters or50,000 Liters.

In the present invention, growth, culturing, and/or fermentation of thetransformed host cells is performed within a temperature rangepermitting survival of the host cells, preferably a temperature withinthe range of about 4° C. to about 55° C., inclusive.

Cell Density

An additional advantage in using Pseudomonas fluorescens in expressingrecombinant proteins includes the ability of Pseudomonas fluorescens tobe grown in high cell densities compared to E.coli or other bacterialexpression systems. To this end, Pseudomonas fluorescens expressionssystems according to the present invention can provide a cell density ofabout 20 g/L or more. The Pseudomonas fluorescens expressions systemsaccording to the present invention can likewise provide a cell densityof at least about 70 g/L, as stated in terms of biomass per volume, thebiomass being measured as dry cell weight.

In one embodiment, the cell density will be at least 20 g/L. In anotherembodiment, the cell density will be at least 25 g/L, 30 g/L, 35 g/L, 40g/L, 45 g/L, 50 g/L, 60 g/L, 70 g/L 80 g/L, 90 g/L., 100 g/L, 110 g/L,120 g/L, 130 g/L, 140 g/L, or at least 150 g/L.

In another embodiments, the cell density at induction will be between 20g/L and 150 g/L;, 20 g/L and 120 g/L; 20 g/L and 80 g/L; 25 g/L and 80g/L; 30 g/L and 80 g/L; 35 g/L and 80 g/L; 40 g/L and 80 g/L; 45 g/L and80 g/L; 50 g/L and 80 g/L; 50 g/L and 75 g/L; 50 g/L and 70 g/L; 40 g/Land 80 g/L.

Expression Levels of Recombinant Protein

The expression systems according to the present invention can expresstransgenic polypeptides at a level at between 5% and 80% total cellprotein (%tcp). In one embodiment, the expression level will be at orabove 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%,70%, 75%, or 80% tcp.

Isolation and Purification

The recombinant proteins produced according to this invention may beisolated and purified to substantial purity by standard techniques wellknown in the art, including, but not limited to, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, nickel chromatography,hydroxylapatite chromatography, reverse phase chromatography, lectinchromatography, preparative electrophoresis, detergent solubilization,selective precipitation with such substances as column chromatography,immunopurification methods, and others. For example, proteins havingestablished molecular adhesion properties can be reversibly fused aligand. With the appropriate ligand, the protein can be selectivelyadsorbed to a purification column and then freed from the column in arelatively pure form. The fused protein is then removed by enzymaticactivity. In addition, protein can be purified using immunoaffinitycolumns or Ni-NTA columns. General techniques are further described in,for example, R. Scopes, Protein Purification: Principles and Practice,Springer-Verlag: N.Y. (1982); Deutscher, Guide to Protein Purification,Academic Press (1990); U.S. Pat. No. 4,511,503; S. Roe, ProteinPurification Techniques: A Practical Approach (Practical ApproachSeries), Oxford Press (2001); D. Bollag, et al., Protein Methods,Wiley-Lisa, Inc. (1996); A K Patra et al., Protein Expr Purif, 18(2): p/182-92 (2000); and R. Mukhija, et al., Gene 165(2): p. 303-6 (1995). Seealso, for example, Ausubel, et al. (1987 and periodic supplements);Deutscher (1990) “Guide to Protein Purification,” Methods in Enzymologyvol. 182, and other volumes in this series; Coligan, et al. (1996 andperiodic Supplements) Current Protocols in Protein Science Wiley/Greene,NY; and manufacturer's literature on use of protein purificationproducts, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See also, for example.,Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, PlenumPress, NY; and Crowe, et al. (1992) QIAexpress: The High LevelExpression & Protein Purification System QUIAGEN, Inc., Chatsworth,Calif.

Detection of the expressed protein is achieved by methods known in theart and includes, for example, radioimmunoassays, Western blottingtechniques or immunoprecipitation.

The recombinantly produced and expressed enzyme can be recovered andpurified from the recombinant cell cultures by numerous methods, forexample, high performance liquid chromatography (HPLC) can be employedfor final purification steps, as necessary.

Certain proteins expressed in this invention may form insolubleaggregates (“inclusion bodies”). Several protocols are suitable forpurification of proteins from inclusion bodies. For example,purification of inclusion bodies typically involves the extraction,separation and/or purification of inclusion bodies by disruption of thehost cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cellsuspension is typically lysed using 2-3 passages through a French Press.The cell suspension can also be homogenized using a Polytron (BrinknanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies can be solubilized, and the lysedcell suspension typically can be centrifuged to remove unwantedinsoluble matter. Proteins that formed the inclusion bodies may berenatured by dilution or dialysis with a compatible buffer. Suitablesolvents include, but are not limited to urea (from about 4 M to about 8M), formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art.

Alternatively, it is possible to purify the recombinant proteins orpeptides from the host periplasm. After lysis of the host cell, when therecombinant protein is exported into the periplasm of the host cell, theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to those skilled in the art. Toisolate recombinant proteins from the periplasm, for example, thebacterial cells can be centrifuged to form a pellet. The pellet can beresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria can be centrifuged and the pellet can be resuspended inice-cold 5 mM MgSO.sub.4 and kept in an ice bath for approximately 10minutes. The cell suspension can be centrifuged and the supernatantdecanted and saved. The recombinant proteins present in the supernatantcan be separated from the host proteins by standard separationtechniques well known to those of skill in the art.

An initial salt fractionation can separate many of the unwanted hostcell proteins (or proteins derived from the cell culture media) from therecombinant protein of interest. One such example can be ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

The molecular weight of a recombinant protein can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture can be ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration can then be ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Recombinant proteins can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

Renaturation and Refolding

Insoluble protein can be renatured or refolded to generate secondary andtertiary protein structure conformation. Protein refolding steps can beused, as necessary, in completing configuration of the recombinantproduct. Refolding and renaturation can be accomplished using an agentthat is known in the art to promote dissociation/association ofproteins. For example, the protein can be incubated with dithiothreitolfollowed by incubation with oxidized glutathione disodium salt followedby incubation with a buffer containing a refolding agent such as urea.

Recombinant protein can also be renatured, for example, by dialyzing itagainst phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 bufferplus 200 mM NaCl. Alternatively, the protein can be refolded whileimmobilized on a column, such as the Ni NTA column by using a linear6M-7M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4,containing protease inhibitors. The renaturation can be performed over aperiod of 1.5 hours or more. After renaturation the proteins can beeluted by the addition of 250 mM immidazole. Immidazole can be removedby a final dialyzing step against PBS or 50 mM sodium acetate pH 6buffer plus 200 mM NaCl. The purified protein can be stored at 4.degree.C. or frozen at -80.degree. C.

Other methods include, for example, those that may be described in M HLee et al., Protein Expr. Purif., 25(1): p. 166-73 (2002), W. K. Cho etal., J. Biotechnology, 77(2-3): p. 169-78 (2000), Ausubel, et al. (1987and periodic supplements), Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries, Coligan, et al. (1996 and periodic Supplements) CurrentProtocols in Protein Science Wiley/Greene, NY, S. Roe, ProteinPurification Techniques: A Practical Approach (Practical ApproachSeries), Oxford Press (2001); D. Bollag, et al., Protein Methods,Wiley-Lisa, Inc. (1996).

VI. Recombinant Polypeptides

The present invention provides improved protein production in bacterialexpression systems. Examples of recombinant polypeptides that can beused in the present invention include polypeptides derived fromprokaryotic and eukaryotic organisms. Such organisms include organismsfrom the domain Archea, Bacteria, Eukarya, including organisms from theKingdom Protista, Fungi, Plantae, and Animalia.

Types of proteins that can be utilized in the present invention includenon-limiting examples such as enzymes, which are responsible forcatalyzing the thousands of chemical reactions of the living cell;keratin, elastin, and collagen, which are important types of structural,or support, proteins; hemoglobin and other gas transport proteins;ovalbumin, casein, and other nutrient molecules; antibodies, which aremolecules of the immune system; protein hormones, which regulatemetabolism; and proteins that perform mechanical work, such as actin andmyosin, the contractile muscle proteins.

Other specific non-limiting polypeptides include molecules such as,e.g., renin, a growth hormone, including human growth hormone; bovinegrowth hormone; growth hormone releasing factor; parathyroid hormone;thyroid stimulating hormone; lipoproteins; alpha. 1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; thrombopoietin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnaturietic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; a serum albumin such as humanserum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; Dnase; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; integrin; protein A or D; rheumatoid factors; a neurotrophicfactor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor suchas NGF-.beta.; cardiotrophins (cardiac hypertrophy factor) such ascardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, orTGF-.beta.5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; anti-HER-2antibody; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides.

The recombinant peptides to be expressed by according to the presentinvention can be expressed from polynucleotides in which the targetpolypeptide coding sequence is operably attached to transcription andtranslation regulatory elements to form a functional gene from which thehost cell can express the protein or peptide. The coding sequence can bea native coding sequence for the target polypeptide, if available, butwill more preferably be a coding sequence that has been selected,improved, or optimized for use in the selected expression host cell: forexample, by synthesizing the gene to reflect the codon use bias of aPseudomonas species such as Pseudomonas fluorescens. The gene(s) thatresult will have been constructed within or will be inserted into one ormore vector, which will then be transformed into the expression hostcell. Nucleic acid or a polynucleotide said to be provided in an“expressible form” means nucleic acid or a polynucleotide that containsat least one gene that can be expressed by the selected bacterialexpression host cell.

Extensive sequence information required for molecular genetics andgenetic engineering techniques is widely publicly available. Access tocomplete nucleotide sequences of mammalian, as well as human, genes,cDNA sequences, amino acid sequences and genomes can be obtained fromGenBank at the URL address http://www.ncbi.nlm.nih.gov/Entrez.Additional information can also be obtained from GeneCards, anelectronic encyclopedia integrating information about genes and theirproducts and biomedical applications from the Weizmann Institute ofScience Genome and Bioinformatics(http:/Ibioinformatics.weizmann.ac.il/cards/), nucleotide sequenceinformation can be also obtained from the EMBL Nucleotide SequenceDatabase ( http://www.ebi.ac.uk/embl/) or the DNA Databank or Japan(DDBJ, http://www.ddbi.nig.ac.jp/; additional sites for information onamino acid sequences include Georgetown's protein information resourcewebsite (http://www-nbrf.georgetown.edu/pir/) and Swiss-Prot(http://au.expasy.org/sprot/sprot-top.html).

EXAMPLES Example 1 Construction of a pyrF Selection Marker System in aP. fluorescens Host Cell Expression System

Reagents were acquired from Sigma-Aldrich (St. Louis Mo.) unlessotherwise noted. LB is 10 g/L tryptone, 5 g/L yeast extract and 5 g/LNaCI in a gelatin capsule (BIO 101). When required, uracil (from BIO101,Carlsbad Calif.) or L-proline was added to a final concentration of 250ug/mL, and tetracycline was added to 15 ug/mL. LB/5-FOA plates containLB with 250 mM uracil and 0.5 mg/mL 5-fluoroorotic acid (5-FOA). M9media consists of 6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 1 g/L NH₄Cl, 0.5 g/LNaCl, 10 mM MgSO₄, 1× HoLe Trace Element Solution, pH7. Glucose wasadded to a final concentration of 1%. The 1000× HoLe Trace ElementSolution is 2.85 g/L H₃BO₃, 1.8 g/L MnCl₂ . 4H2O, 1.77 g/L sodiumtartrate, 1.36 g/L FeSO₄. 7H2O, 0.04 g/L CoCl₂. 6H₂O, 0.027 g/L CuCl₂.2H₂O, 0.025 g/L Na₂MoO₄. 2H₂O, 0.02 g/L ZnCl₂.

Oligonucleotides Used Herein

MB214pyrF1 (NotI site in bold) 5′-GCGGCCGCTTTGGCGCTTCGTTTACAGG-3′ (SEQID NO: 14) MB214pyrR1 (PvuI site in bold; KpnI site in underlined bold)5′-CGATCGG GTACC TGTCGAAGGGCTGGAGACA (SEQ ID NO: 15) T-3′ pyrFPstF (PstIsite in bold) 5′-AACTGCAGGATCAGTTGCGGAGCCTTGG-3′ (SEQ ID NO: 16)pyrFoverlap 5′-TGCTCACTCTAAAAATCTGGAATGGGCTCTC (SEQ ID NO: 17) AGGC-3′pyrFXbaR2 (XbaI site in bold) 5′-GCTCTAGATGCGTGGCTGGATGAATGAA-3′ (SEQ IDNO: 18) pyrana1F 5′-GGCGTCGAACAGGTAGCCTT-3′ (SEQ ID NO: 19) pyrana1R5′-CTCGCCTCCTGCCACATCAA-3′ (SEQ ID NO: 20) M13F(−40)5′-CAGGGTTTTCCCAGTCACGA-3′ (SEQ ID NO: 21)

Cloning of a pyrF gene from P. fluorescens

The pyrF gene was cloned from P. fluorescens by polymerase chainreaction (PCR) amplification, using primers MB214pyrF1 and MB214pyrR1that bind 297 bp upstream from the pyrF gene start codon and 212 bpdownstream of its stop codon, respectively. Restriction sites wereincluded at the 5′ ends of the primers to facilitate further cloningreactions The amplified region upstream of the pyrF open reading frame(ORF) was estimated as long enough to include the native promoterupstream of pyrF. A strong stem-loop structure at 14-117 bp downstreamof the pyrF ORF, which may be a transcription terminator, was alsoincluded in the downstream flanking region.

To PCR-amplify the pyrF gene, the high-fidelity PROOFSTART DNApolymerase was mixed in a 50 uL reaction volume containing bufferprovided by the manufacturer (Qiagen, Valencia Calif.) 0.3 mM dNTPs(Promega, Madison, Wis.), 1 uM each of MB214pyrF1 and MB214pyrR1primers, and about 0.3 μg of genomic DNA from P. fluorescens MB214. Theamplification conditions were 5 min at 95° C., followed by 35 cycles ofa 30 sec denaturation at 94° C., 30 sec annealing at 57° C., and a 2 minextension at 72° C., followed by a final step a 72° C. for 10 min. Thereaction was separated on a 1% gel of SEAKEM GTG agarose (fromBioWhittaker Molecular Applications , Rockland Me.). The expected 1.2 kbband was excised from the gel and purified by extraction on aULTRAFREE-DA centrifugal gel nebulizer from Millipore (Bedford Mass.)column and de-salted into Tris-HCI buffer with a MICRoBIoSPIN 6 P-6polyacrylamide spin column (from Bio-Rad, Hercules Calif.).

The cloned gene contained a single ORF, encoding orotidine 5′ phosphatedecarboxylase. The identity of the gene was further confirmed as pyrF byits high similarity (P-value of 3.3×10⁻⁷⁸) along the entire length ofthe gene (209 out of 232 residues) to the pyrF gene from P. aeruginosa,which had been previously reported (Strych et al., 1994). The P.fluorescens strain used was found to contain no other copies of anypyrFgenes.

Sequencing was performed by The Dow Chemical Company. The pyrF sequenceis presented within SEQ ID NO:1.

Construction of a pyrF(−) P. fluorescens

To construct a pyrF(−) P. fluorescens , the cell's genomic pyrF gene wasaltered by deleting of the ORF between and including the gene's startand stop codons. The deletion was made by fusing in vitro the upstreamand downstream regions flanking the pyrF region on a nonreplicatingplasmid, then using allele exchange, i.e. homologous recombination, toreplace the endogenous pyrF gene in MB101 with the deletion allele.

To construct the fusion of the flanking regions, the “Megaprimer” method(Barik 1997) was used, whereby the region upstream and then downstreamof the desired deletion were subsequently amplified by PCR using anoverlapping primer with homology on both sides of the desired deletion,so that the flanking regions become linked, leaving out the pyrF ORF.The upstream region was amplified from MB214 genomic DNA using theProofstart polymerase (Qiagen) as described above, with the primerspyrFPstF and pyrFoverlap, and an extension time of 1 minute. After gelpurification using binding to glass milk (GENECLEAN Spin Kit fromBio101, Carlsbad, Calif., USA), the 1 kB product was used as the“Megaprimer” for the second amplification.

Because there was difficulty amplifying the desired product in thissecond step, a template containing the genomic pyrF region was made byPCR amplification in order to increase the template quantity. HOTSTARTAQDNA polymerase (from Qiagen, Valencia Calif.) was used with P.fluorescens genomic DNA and the pyrFPstF and pyrFXbaR2 primers. TheMegaprimer and the pyrFXbaR2 primer were then used with this templateand HOTSTARTAQ polymerase, to amplify the deletion product by PCR, usingamplification conditions of 15 min at 95° C., followed by 30 cycles of a30 sec denaturation at 94° C., 30 sec annealing at 59° C., and a 2 minextension at 72° C., followed by a final step at 72° C. for 3 min. Theexpected 2 kB band was separated from a number of other products by gelelectrophoresis, and then gel purified as above and cloned into plasmidpCR2.1Topo (from Invitrogen, Carlsbad CA) according to instructions fromthe manufacturer, to form pDOW1215-7 Sequencing the PCR-amplified regionof pDOW1215-7 showed that there were 3 mutations introduced by theamplification process; all three changes were within 112 bp downstreamof the stop codon for pyrF. Sequencing through this area was difficult,because the process of the reaction stopped in this area. Analysis byM-FOLD (GCG) of the secondary structure of RNA that would be encoded bythis area showed the presence of a very stable stem-loop structure and arun of uridine residues that is characteristic of a rho-independenttranscription terminator. None of the mutations occurred in the openreading frame. pDOW 1215-7 was used to delete the chromosomal pyrF genein MB 101. To do this, first, electrocompetent P. fluorescens cells madeaccording to the procedure of Artiguenave et al. (1997), weretransformed with 0.5 μg of the purified plasmid. Transformants wereselected by plating on LB medium with kanamycin at 50 μg/mL. Thisplasmid cannot replicate in P. fluorescens , therefore kanamycinresistant colonies result from the plasmid integrating into thechromosome. The site of integration of the plasmid was analyzed by PCRusing the HOTSTARTAQ polymerase and primers pryanalF and M13F(−40),annealing at 57° C. and with an extension time of 4 min. One out of the10 isolates (MB101::pDOW1215-7#2) contained an insertion of pDOW1215-7into the downstream region (2.8 kB analytical product) and in the othernine were in the upstream region (2.1 kb analytical product).

Second, to identify strains that had lost the integrated plasmid byrecombination between the homologous regions the following analyticalPCR procedure was used: MB1010::pDOW1215-7#2 was inoculated from asingle colony into LB supplemented with 250 mM uracil, grown overnight,and then plated onto LB-uracil and 500 μg/mL 5-fluoroorotic acid(5-FOA—Zymo Research, Orange Calif.). Eight colonies were analyzed byPCR with HOTSTARTAQ and primers pyranalF and pyranalR, annealing at 57°C. and extending for 4 min. The expected size of the amplified productfrom the parent MB 101 was 3.2 kB, or if the pyrF gene was deleted, then2.5 kB. Each of the colonies gave rise to the 2.5 kB band expected froma deletion of pyrF. The first three isolates were purified and namedPFG116, PFG117, and PFG118 (also known as DC36). The three isolatesexhibit the phenotype expected from a pyrF deletion, i.e. they aresensitive to kanamycin, uracil is required for growth, and they areresistant to 5-FOA. The DNA sequence of PFG118 was identical to that ofthe amplified regions in pDOW1215-7; i.e. the three mutations in thestem-loop structure immediately downstream from pyrF were incorporatedinto the PFG 118 genome, along with the pyrF deletion.

Use of the pyrF Gene as a Selection Marker in P. fluorescens ExpressionSystem

The ability of the pyrF gene to act as a selectable marker was tested bycloning it into a pMYC expression plasmid containing both an existingtetracycline resistance marker and the target enzyme coding sequenceunder the control of the tac promoter. For this, the plasmid pMYC5088was digested at 37° C. for 2 hr with SnaBI in a 50 uL reaction using NEBBuffer 4 and 0.1 mg/mL of bovine serum albumen (BSA) (from New EnglandBiolabs, Beverly Mass.). The reaction mixture was then treated at 70° C.for 20 min to inactivate the enzyme, then gel-purified as describedabove. 60 ng of the SnaBI-digested pMYC5088 was ligated to 50 ng of theMB214pyrF1- MB214pyrR1 PCR product using the FAST-LINK DNA Ligation Kit(Epicentre Technologies, Madison Wis.). After 1 hr at 25° C., thereaction was stopped by treating the mixture at 70° C. for 20 min. Theresult was then transformed into chemically-competent JM109 E.coli cells(Promega Corp., Madison Wis.) using conditions recommended by themanufacturer.

Transformants were selected on LB medium containing tetracycline at 15μg/mL. Plasmid DNA was prepared from 12 isolates using the QiaPrep SpinMiniprep Kit (Qiagen, Valencia Calif.) and screened with NotI and EcoRI,which indicated that one isolate contained the desired clone, pDOW1249-2(FIG. 2). The plasmid pDOW1249-2 was transformed into pyrF(−) P.fluorescens containing a pCN plasmid containing a lacI repressorexpression cassette and a kanamycin resistance marker gene. Isolateswere tested in shake flasks and in 20-L fernentors.

Isolates were grown in minimal salts medium and kanamycin, but notetracycline, so that the only selective pressure for the pDOW1249-2plasmid was provided by the ability of the pyrF gene on the plasmid tocomplement the pyrF deletion in the chromosome. As determined bySDS-PAGE analysis, the amount of target protein produced by the newstrain in the shake flask test was similar to that of the controlstrain, a genomically pyrF(+) P. fluorescens control system containingthe same two plasmids, but for the absence of the pyrF gene inpDOW1249-2, and grown on the same medium but further supplemented withtetracycline in order to maintain the plasmid (data not shown). Twostrains were chosen for further analysis at the 20-L scale, based on theamount of target protein seen on the SDS-PAGE gel and OD₅₇₅ values inshake flasks. Both strains showed a level of accumulation of targetprotein within the normal range observed for the control strain (FIG.1).

Example 2 Construction of a pyrF—proC Dual Auxotrophic Selection MarkerSystem in a P. fluorescens Host Cell Expression System

Oligonucleotides Used Herein proC1 5′-ATATGAGCTCCGACCTTGAGTCGGCCATTG-(SEQ ID NO: 22) 3′ proC2 5′-ATATGAGCTCGGATCCAGTACGATCAGCAGG (SEQ ID NO:23) TACAG-3′ proC3 5′-AGCAACACGCGTATTGCCTT-3′ (SEQ ID NO: 24) proC55′-GCCCTTGAGTTGGCACTTCATCG-3′ (SEQ ID NO: 25) proC65′-GATAAACGCGAAGATCGGCGAGATA-3′ (SEQ ID NO: 26) proC75′-CCGAGCATGTTTGATTAGACAGGTCCTTATT (SEQ ID NO: 27) TCGA-3′ proC85′-TGCAACGTGACGCAAGCAGCATCCA-3′ (SEQ ID NO: 28) proC95′-GGAACGATCAGCACAAGCCATGCTA-3′ (SEQ ID NO: 29) genF25′-ATATGAGCTCTGCCGTGATCGAAATCCAGA- (SEQ ID NO: 30) 3′ genR25′-ATATGGATCCCGGCGTTGTGACAATTTACC- (SEQ ID NO: 31) 3′ XbaNotDraU2 linker5′-TCTAGAGCGGCCGCGTT-3′ (SEQ ID NO: 32) XbaNotDraL linker5′-GCGGCCGCTCTAGAAAC-3′ (SEQ ID NO: 33)

Cloning of proC from P. fluorescens and Formation of a pCN ExpressionPlasmid Containing proC

Replacing Antibiotic Resistant Gene in pCN51lacI with proC

The proC ORF and about 100 bp of adjacent upstream and downstreamsequence was amplified from MB101 genomic DNA using proC1 and proC2, anannealing temperature of 56° C. and a 1 min extension. After gelpurification of the 1 kB product and digestion with SacI, the fragmentwas cloned into SacI-digested pDOW1243 (a plasmid derived from pCN51lacIby addition of a polylinker and replacement of kanR with the gentamycinresistance gene), to create pDOW1264-2. This plasmid was tested in theproC(−) mutant strain PFG932 for its ability to regulate amylasesynthesis from pDOW1249-2. Expressed target enzyme production levels atthe 20-L scale was similar to that of the dual-antibiotic-resistancemarker control strain DC88 (data not shown).

The genR antibiotic marker gene was then removed from the pDOW 1264-2(FIG. 3) to create an antibiotic-marker-free plasmid with proC and lacI.Removing the genR gene was accomplished by restriction digestion ofpDOW1264-2 with BamHI, purification of the 6.1 kB fragment, ligation toitself, and electroporation into the proC(−) P. fluorescens hostPFG1016. Isolates were checked by restriction digestion using EcoRI. Theresulting plasmid was named pDOW1306-6. Analytical restriction digestswith EcoRI and sequencing across the BamHI junction verified theidentity of the plasmid and the proper orientations of the genestherein.

Sequencing was performed by The Dow Chemical Company. The proC sequenceis presented within SEQ ID NO:4.

Construction of Target Enzyme Expression Plasmid Containing a pyrFMarker in Place of an Antibiotic Resistance Marker

The antibiotic-marker-free production plasmid, pDOW1269-2, containing atarget enzyme-encoding gene under control of a tac promoter, wasconstructed by restriction digestion of pDOW1249-2 with PvuI to removethe tetR/tetA genes. Derived from pMYC5088 by insertion of the pyrF genefrom MB214, pDOW1249-2 was prepared as described in Example 1. The 10.6kB PvuI fragment was gel-purified, ligated to itself, transformed intoPFG118/pCN51lacI by electroporation and spread on M9 glucose mediumcontaining kanamycin (to retain the pCN51lacI). Plasmid DNA was isolatedand analytical restriction digests with NcoI were carried out; twoisolates showed a restriction digest that was consistent with theexpected bands. Both isolates were sequenced across the PvuI junction,which verified the identity of the plasmids and the proper orientationsof the genes therein.

Construction of a Pseudomonas fluorescens Strain with Genomic Deletionsof pyrF and proC

PFG118, a P. fluorescens MB 101 strain with a deletion of pyrF, wasdescribed in Example 1.

Construction of pDOW 1261-2, a Vector for Gene Replacement and Deletion

The vector pDOW1261-2 was designed to create clean deletions of genomicDNA, using marker exchange by the cross-in/cross-out method (Toder 1994;Davison 2002), by combining the following properties:

-   -   a ColEI replication origin that functions only in E.coli and not        in P. fluorescens;    -   a selectable marker (tetR/tetA) for integration of the plasmid        into the chromosome;    -   a counterselectable marker (pyrF) that allows for selection for        loss of the inserted plasmid (as long as the host strain is        pyrF-); cells that lose the pyrF gene are resistant to 5-FOA;        and    -   a blunt-end cloning site, SrfI, which has an uncommon 8 bp        recognition site - if the desired insert lacks the site, the        efficiency of insertion can be increased by adding SrfI        (Stratagene, La Jolla Calif.) to the ligation reaction to        re-cleave vectors that ligate without an insert.

To construct this vector, a 5 kB PstI to EcoRI fragment containing thetetR, tetA, and pyrF genes was cloned into pCRScriptCAM (Stratagene, LaJolla Calif.) that had been digested with PstI and EcoRI, creating pDOW1261-2.

Construction of a Vector to Delete proC from the Chromosome

To construct a deletion of proC, the copies of the flanking regionsupstream and downstream of the proC gene were joined together by PCR,and then cloned into the pDOW1261-2 gene replacement vector. The proC7primer, which bridges the proC ORF, was designed to delete the entirecoding sequence from the ATG start codon to the TAG stop codon. Anadditional 16 bp downstream of the stop codon was also included in thedeletion.

To make the PCR fusion of regions upstream and downstream from proC, theMegaprimer method of PCR amplification was used (Barik 1997). To makethe megaprimer, the 0.5 kB region directly upstream of the proC openreading frame was amplified by PCR from MB214 genomic DNA, using primersproC5 and proC7. Primer proC7 overlaps the regions upstream anddownstream of the proC ORF. The polymerase chain reaction was carriedout with 1 uM of primers, 200 uM each of the four dNTPs, and Herculasehigh-fidelity polymerase (Stratagene, La Jolla Calif.) in the bufferrecommended by the vendor. Herculase is a high-fidelity enzyme thatconsists mostly of Pfu polymerase, which leaves blunt ends. Theamplification program was 95° C. for 2 min, 30 cycles of 95° C. for 30sec, 50° C. for 30 sec, and 72° C. for 1 min per kB, followed by 10 minat 72° C. The amplified products were separated by 1% agarose gelelectrophoresis in TBE and visualized using ethidium bromide. A gelslice containing the DNA was cut from the gel and purified as above The1.3 kB region downstream from the proC gene was amplified using primersproC3 and proC6, to serve as a template for subsequent reactions. Thesame amplification protocol was used, except for an annealingtemperature of 60° C. The reaction was checked on an agarose gel, andthen purified using the StrataPrep PCR Purification Kit (Stratagene, LaJolla Calif.).

In the second step to make the deletion fusion, the megaprimer was usedas one of the primers in a PCR reaction along with primer proC6, andwith the proC3-proC6 PCR reaction as the template. An annealingtemperature of 61° C. and extension time of 2 min was used. The 1 kB PCRproduct was purified and blunt-end ligated into the suicide vectorpDOW1261-2 that had been digested with SrfI. SrfI was included in theligation in order to decrease background caused by re-ligation of thevector, as according to instructions from the manufacturer (pCRScriptCamCloning Kit—Stratagene, La Jolla Calif.). The ligation was transformedinto DH10 β (Gibco BRL Life Technologies, now Invitrogen, CarlsbadCalif.) by electroporation (2 mM gap cuvette, 25 μF, 2.25 kV, 200 Ohms)(Artiguenave et al. 1997), and isolates were screened using the DraIIIrestriction enzyme. The PCR amplified region of each isolate wassequenced by The Dow Chemical Company; isolate pDOW1305-6 was verifiedas containing the correct genomic DNA sequence.

Formation of the P. fluorescens pvrF-proC Double Deletion

To make a doubly deleted strain, PFG118 was transformed with pDOW1305-6by electroporation as described above. Analytical PCR on the colonieswith primers proC8 and the M13/pUC Reverse Sequencing Primer (−48)(which hybridizes to the plasmid only) (New England Biolabs, BeverlyMass.), using HotStarTaq (Qiagen, Valencia Calif.), an annealingtemperature of 59° C. and an extension time of 4 min, showed that 9 outof 22 isolates had the plasmid integrated into the region upstream fromproC, and 7 out of 22 had the plasmid integrated downstream (data notshown). Three of each orientation were purified to single colonies. Thethree isolates PFG118::1305-6.1, -6.8, -6.10 have an insertion in theregion upstream, and the three isolates PFG118::1305-6.2, -6.3, -6.9have an insertion in the region downstream.

To select for cells that have carried out a homologous recombinationbetween the plasmid and the chromosome genes thereby leaving a deletion,PFG118::1305-6.1 and -6.2 were grown to stationary phase in 50 mL of LBwith uracil and proline supplementation and then plated on LB-5-FOA withuracil and proline supplementation. Cells that lose the integratedplasmid by recombination also lose the pyrF gene, and are thereforeexpected to be resistant to 5-FOA which would otherwise be convertedinto a toxic compound. PCR analysis with proC8 and proC9 was carried outto distinguish between cells that had lost the plasmid and regeneratedthe original sequence, and those that had left the deletion. Twoisolates with the expected 1.3 kB band were chosen from each of the twoselections and named PFG1013, PFG1014, PFG1015 and PFG1016 (also knownas DC164). All four isolates were unable to grow on M9 glucose unlessboth proline and uracil were added, and were tetracycline-sensitive. Thegenomic region of PFG118 (wild type proC) and PFG1016 (proC deletion)was amplified by PCR (primers proC8 and proC9, HotStarTaq polymerase,63° C. annealing and 3 min extension) and sequenced. The region betweenproC5 and proC6 of strain PFG1016 was identical to the parent, exceptfor the expected 835 bp deletion.

Construction of a Dual Auxotrophic Selection Marker Expression SystemPFG 1016/pDOW 1306-6 pDOW 1269-2

Plasmids were isolated from strain PFG118 pCN51lacI pDOW1269-2 byHISPEED Plasmid Midi Kit (Qiagen, Valencia Calif.). The pDOW1269-2 waspartially purified from the pCN51lacI by agarose gel electrophoresis andthen electroporated into PFG1016 pDOW1306-6 . Transformants wereselected on M9/glucose without supplementation. Because there was apossibility that some of the pCN51lacI contaminating the pDOW1269-2preparation would also be cotransformed into the cells, three isolatesfrom each transformation were tested for sensitivity to kanamycin, theantibiotic marker carried on pCN51lac; all six were found sensitive. Allsix strains were found to express the target enzyme, in a test of targetenzyme activity. PCR analysis showed that all six also contained thechromosomal proC deletion.

Restriction digestion of plasmids isolated from the transformants wasconsistent with the expected pattern.

Performance Testing of the Dual Auxotrophic Marker Expression System inShake Flasks

The six strains were then tested in shake flasks as described above inExample 1. Induction of target enzyme expression was initiated at 26hours by addition of IPTG. The OD₅₇₅ for all six strains was comparableto that of the dual-antibiotic-resistance marker expression systemcontrol, DC88. Target enzyme production levels in all six were alsocomparable to that of the control, as assessed by SDS-PAGE. The twostrains that achieved the highest OD₅₇₅, strains 1046 and 1048, wereselected for further characterization.

Performance Testing of the Dual Auxotrophic Marker Expression System in20-L Bioreactors

Strains 1046 and 1048 were subsequently tested in 20-L bioreactors.Induction of target enzyme expression was initiated at 26 hours byaddition of IPTG. Both strains achieved performance levels within thenormal range for the DC88 control strain, for both OD₅₇₅ and targetenzyme activity. The performance averages of these two strains are shownin FIG. 1. Restriction digests of plasmids prepared from samples takenat the seed stage and at a time just before the 26-hour start ofinduction showed a pattern consistent with that expected. Analytical PCRof genomic DNA carried out on the same samples demonstrated theretention of the proC deletion and the pyrF deletion. Aliquots of the 25hr samples were plated on tetracycline-, gentamycin-, orkanamycin-supplemented media; no cell growth was observed, thusconfirming the absence of antibiotic resistance gene activity.

Analysis of strain 1046 (also known as DC167) in 20-L bioreactors wasrepeated twice with similar results. Plasmid stability at the seed stageand after 25 hours of fermentation (immediately before induction) wastested by replica plating from samples that had been diluted and platedon complete media. Both plasmids were present in more than 97% of thecolonies examined, indicating the lack of cross feeding revertants ableto survive without the plasmid and the stable maintenance of theexpression vector in Pseudomonas fluorescens.

Results

Both of the pyrF expression systems performed as well as the controlsystem in which only antibiotic resistance markers were used (FIG. 1).For the control strain, there is no negative effect of cross-feeding,since any importation of exogenous metabolites from lysed cells does notdecrease or remove the selection pressures provided by the antibioticsin the medium. The expected decreases in performance expected as aresult of cross-feeding on the two pyrF expression systems weresurprisingly not observed.

Example 3 Chromosomal Integration of lacI. lacI^(Q) and lacI^(Q1) in P.fluorescens

Three P. fluorescens strains have been constructed, each with one ofthree different Escherichia coli lacI alleles, lacI (SEQ ID NO:9),lacI^(Q) (SEQ ID NO: 11), and lacI^(Q1) (SEQ ID NO:12), integrated intothe chromosome. The three strains exhibit differing amounts of LacIrepressor accumulation. Each strain carries a single copy of its lacIgene at the levansucrase locus (SEQ ID NO:13) of P. fluorescens DC36,which is an MB101 derivative (see TD Landry et al., “Safety evaluationof an α-amylase enzyme preparation derived from the archaeal orderThermococcales as expressed in Pseudomonas fluorescens biovar I,”Regulatory Toxicology and Pharmacology 37(1): 149-168(2003)) formed bydeleting the pyrF gene thereof, as described above.

No vector or other foreign DNA sequences remain in the strains. Thestrains are antibiotic-resistance-gene free and also contain a pyrFdeletion, permitting maintenance, during growth in uracilun-supplemented media, of an expression plasmid carrying a pyrF+ gene.Protein production is completely free of antibiotic resistance genes andantibiotics.

MB214 contains the lacI-lacZYA chromosomal insert described in U.S. Pat.No. 5,169,760. MB214 also contains a duplication in the C-terminus ofthe LacI protein, adding about 10 kDa to the molecular weight of theLacI repressor.

Construction of Vector pDOW1266-1 for Insertion of Genes into theLevansucrase Locus

Plasmid pDOW1266-1 was constructed by PCR amplification of the regionupstream of and within the P. fluorescens gene for levansucrase (SEQ IDNO:13), replacing the start codon with an XbaI site, using theMegaprimer method, see A Barik, “Mutagenesis and Gene Fusion byMegaprimer PCR,” in BA White, PCR Cloning Protocols 173-182 (1997)(Humana). PCR was performed using HERCULASE polymerase (Stratagene,Madison Wis., USA) using primers LEV1 (SEQ ID NO:34) and LEV2 (SEQ IDNO:35), and P. fluorescens MB214 genomic DNA as a template (see belowfor oligonucleotide sequences). Primer LEV2 (SEQ ID NO:35) contains thesequence that inserts an XbaI site. The reaction was carried out at 95°C. for 2 min, 35 cycles of [95° C. for 30 sec, 58° C. for 30 sec, 72° C.for 1 min], followed by 10 min at 72° C. The 1 kB product was gelpurified and used as one of the primers in the next reaction, along withLEV3 (SEQ ID NO:36), using MB214 genomic DNA as a template and the sameconditions except that extension time was 2 min. The 2 kB product wasgel purified and re-amplified with LEV2 (SEQ ID NO: 35) and LEV3 (SEQ IDNO. 36) in order to increase the quantity.

Oligonucleotides Used LEV1 5′-TTCGAAGGGGTGCTTTTTCTAGAAGTAAGTC (SEQ IDNO: 34) TCGTCCATGA LEV2 5′-CGCAAGGTCAGGTACAACAC (SEQ ID NO: 35) LEV35′-TACCAGACCAGAGCCGTTCA (SEQ ID NO: 36) LEV7 5′-CTACCCAGAACGAAGATCAG(SEQ ID NO: 37) LEV8 5′-GACTCAACTCAATGGTGCAGG (SEQ ID NO: 38) BglXbaLacF5′-AGATCTCTAGAGAAGGCGAAGCGGCATGCAT (SEQ ID NO: 39) TTACG lacIR45′-ATATTCTAGAGACAACTCGCGCTAACTTACA (SEQ ID NO: 40) TTAATTGC Lacpro95′-ATATTCTAGAATGGTGCAAAACCTTTCGCGG (SEQ ID NO: 41) TATGGCATGA LacIQF5′-GCTCTAGAAGCGGCATGCATTTACGTTGACA (SEQ ID NO: 42) CC LacINXR5′-AGCTAGCTCTAGAAAGTTGGGTAACGCCAGG (SEQ ID NO: 43) GT lacIQ15′-AGTAAGCGGCCGCAGCGGCATGCATTTACGT (SEQ ID NO: 44)TGACACCACCTTTCGCGGTATGGCATG The Oligos Below were Used for AnalyticalSequencing Only lacIF1 5′-ACAATCTTCTCGCGCAACGC (SEQ ID NO: 45) lacIF25′-ATGTTATATCCCGCCGTTAA (SEQ ID NO: 46) lacIR1 5′-CCGCTATCGGCTGAATTTGA(SEQ ID NO: 47) lacIR2 5′-TGTAATTCAGCTCCGCCATC (SEQ ID NO: 48) SeqLev5AS5′-TATCGAGATGCTGCAGCCTC (SEQ ID NO: 49) SeqLev3S 5′-ACACCTTCACCTACGCCGAC(SEQ ID NO: 50) LEV10 5′-TCTACTTCGCCTTGCTCGTT (SEQ ID NO: 51)

The LEV2 - LEV3 amplification product was cloned into the SrfI site ofpDOW1261-2, a suicide vector that contains P. fluorescens pyrF+ gene asa selection marker to facilitate selection for cross-outs. The newplasmid was named pDOW1266-1. The amplified region was sequenced.

Cloning the lacI Genes into Insertion Vector pDOW1266-1

The E.coli lacI gene was amplified from pCN51lacI with primersBglXbaLacF (SEQ ID 10 NO:39) and lacIR4 (SEQ ID NO. 40), using HERCULASEpolymerase (annealing at 62° C. and extension time of 2 min). After gelpurification and digestion with XbaI, the lacI gene was cloned into theXbaI site of pDOW1266-1, to make pDOW1310. The lacI^(Q) gene was createdby PCR amplification using pCN51lacI as a template with 15 primerslacpro9 (SEQ ID NO. 41) and lacIR4 (SEQ ID NO. 40), using HERCULASEpolymerase (annealing at 62° C. and extension time of 2 min). After gelpurification and digestion with XbaI, it was cloned into the XbaI siteof pDOW1266-1, to make pDOW1311.

The lacI^(Q1) gene was created by amplifying the lacI gene from E.coliK12 (ATCC47076) using primers lacIQ1 (SEQ ID NO. 44) and lacINXR (SEQ IDNO. 43) and cloned into pCR2.1 Topo (Invitrogen, Carlsbad, Calif., USA),to make pCR2-lacIQ1. The lacI^(Q1) gene was reamplified from pCR2-lacIQ1using primers lacIQF (SEQ ID NO. 42) and lacINXR (SEQ ID NO. 43) withHerculase polymerase (61° C. annealing, 3 min extension time, 35cycles). After gel purification and digestion with XbaI, the PCR productwas cloned into the XbaI site of pDOW1266-1, to make pDOW1309.

The PCR amplified inserts in pCR2-lacIQ1, pDOW1310, pDOW1311, andpDOW1309 were sequenced (using primers lacIF1 (SEQ ID NO:45), lacIF2(SEQ ID NO. 46), lacIR1 (SEQ ID NO. 47), lacIR2 (SEQ ID NO. 48),SeqLev5AS (SEQ ID NO. 49), SeqLev3S (SEQ ID NO. 50), and LEV10 (SEQ IDNO. 51)) to insure that no mutations had been introduced by the PCRreaction. In each case, an orientation was chosen in which the lacI wastranscribed in the same direction as the levansucrase gene. Although thelevansucrase promoter is potentially able to control transcription oflacI, the promoter would only be active in the presence of sucrose,which is absent in the fermentation conditions used.

Construction of P. fluorescens Strains with Integrated lacI Genes at theLevansucrase Locus

The vectors pDOW1309, pDOW1310, and pDOW1311 were introduced into DC36by electroporation, first selecting for integration of the vector intothe genome with tetracycline resistance. Colonies were screened todetermine that the vector had integrated at the levansucrase locus byPCR with primers LEV7 (SEQ ID NO. 37) and M13R (from New EnglandBiolabs, Gloucester Mass., USA). To select for the second cross-overwhich would leave the lacI gene in the genome, the isolates were grownin the presence of 5′-fluoroorotic acid and in the absence oftetracycline. Recombination between the duplicated regions in the genomeeither restores the parental genotype, or leaves the lacI gene. Theresulting isolates were screened for sensitivity to tetracycline, growthin the absence of uracil, and by PCR with primers LEV7 (SEQ ID NO. 37)and LEV8 (SEQ ID NO. 38). The names of the new strains are shown inTable 17. To obtain sequence information for genomic regions, PCRproducts were sequenced directly, see E Werle, “Direct sequencing ofpolymerase chain reaction products, ”Laboratory Methods for theDetection of Mutations and Polymorphisms in DNA 163-174 (1997). For eachstrain, the sequencing confirmed the identity of the promoter, theorientation of the lacI variant relative to the flanking regions, andwhether there were any point mutations relative to the parentalsequence. The sequences of DC202 and DC206 were as expected. Thesequence of DC204 showed a point mutation within the levansucrase openreading frame, downstream of lacl^(Q), which did not change any codingsequence and therefore is inconsequential. TABLE 17 P. FLUORESCENSSTRAINS WITH LACI ALLELES INTEGRATED INTO THE GENOME Plasmid used tomake Strain Designation the lacI insertion Genotype DC202 pDOW1310-1pyrF lev::lacI DC204 pDOW1311-4 pyrF lev::lacI^(Q) DC206 pDOW1309oriApyrF lev::lacI^(Q1)

Analysis of Relative Concentration of LacI in the lacI Integrants,Compared to pCN51lacI

UnBlot is a method analogous to Western analysis, in which proteins aredetected in the gel without the need for transfer to a filter. Thetechnique was carried out following the directions from PierceBiotechnology (Rockford, Ill., USA), the manufacturer. Analysis usingUnBlot showed that the amount of LacI in each of the new integrantstrains was higher than in MB214. MB214 contains the lacI-lacZYA insertdescribed in U.S. Pat. 5,169,760. The relative concentration of LacI inthe lacI^(Q) and lacI^(Q1) integrants was about the same as in strainscarrying pCN51lacI, the multi-copy plasmid containing lacI. See FIG. 5.

A dilution series was carried out in order to assess more precisely therelative difference in LacI concentration in MB214, DC202 (lacIintegrated) and DC206 (lacI^(Q1) integrated). MB101pCN51lacI, DC204 andDC206 have about 100 times more LacI than MB214, whereas DC202 has about5 times more.

Example 4 Nitrilase Expression and Transcription

Strain DC140 was constructed by introducing into P. fluorescens MB214 atetracycline- resistant broad-host-range plasmid, pMYC1803 (WO2003/068926), into which a nitrilase gene (G DeSanthis et al., J Amer.Chem. Soc. 125:11476-77 (2003)), under the control of the Ptac promoter,had been inserted. In order to compare regulation of un-inducedexpression of the target gene in DC202 and DC206 with MB214, the samenitrilase gene was cloned onto a pMYC1803 derivative where thetetracycline-resistance gene has been replaced by a pyrF selectionmarker. The new plasmid, pDOW2415, was then electroporated into DC202and DC204, resulting in DC239 and DC240, respectively. DC140, DC239 andDC240 were cultured in 20 L fermentors by growth in a mineral saltsmedium fed with glucose or glycerol, ultimately to cell densitiesproviding biomasses within the range of about 20 g/L to more than 70 g/Ldry cell weight (See WO 2003/068926). The gratuitous inducer of the Ptacpromoter, IPTG, was added to induce expression.

The ratio of pre-induction nitrilase activities of DC140 to DC239 toDC240 was 6:2:1. RNA analysis by Northern blots of the same samplesrevealed the same ranking of derepression. Based on densitometricmeasurements, the ratio of un-induced transcript levels ofDC140:DC239:DC240 was 2.4:1.4:1.0. Shortly after induction (30 min) with0.3mM IPTG, the levels of transcript of all the strains were the same.Post-induction nitrilase productivity rates were also comparable. Thisindicated that the concentration of inducer used was sufficient to fullyinduce the Ptac promoter in these three strains despite their differentLacI protein levels. However, fermentations of the most derepressedstrain, DC140, suffered significant cell lysis accompanied with loss ofnitrilase activity after approximately 24 hours post-induction.Induction of the improved, more tightly regulated strains, DC239 andDC240, could be extended to more than 48 hours post induction, whilemaintaining high nitrilase productivity, with the ultimate result of adoubling of nitrilase yields. See FIG. 6.

Results

The examples indicate It that the use of a LacI-encoding gene other thanas part of a whole or truncated Plac-lacI-lacZYA operon in Pseudomonadsresulted in substantially improved repression of pre-inductionrecombinant protein expression, higher cell densities incommercial-scale fermentation, and higher yields of the desired productin comparison with previously taught lacI-lacZYA Pseudomonad chromosomalinsertion (U.S. Pat. No. 5,169,760). The results also indicated that thelacI insertion is as effective in producing LacI repressor protein inPseudomonas fluorescens , thereby eliminating the need to maintain aseparate plasmid encoding a LacI repressor protein in the cell andreducing potential production inefficiencies caused by such maintenance.

In addition to being antibiotic free, derepression during the growthstage in DC239 and DC240 was up to 10 fold less than the MB214 strain.Pre-induction nitrilase activity levels of DC239 and DC240 averaged 0.4U/ml in more than 13 separate fermentations, and cell density andnitrilase expression in DC239 and DC240 did not decay during extendedinduction phase, as it did in DC140. Given the higher derepression,DC239 and DC240 fermentation runs decreased the time of the growth phaseby more than 30%, reducing fermentation costs.

Example 5 Construction of tac Promoter with a Single Optimal lacOperator and with Two lac Operators

The native tac promoter only has a single native lac operator,AATTGTGAGCGGATAACAATT, at the O1 position (FIG. 4). In the firstconstruct, pDOW1418, the native operator was replaced by the moresymmetrical lacOid operator sequence 5′-AATTGTGAGC GCTCACAATT - 3′ (SEQ.ID. NO. 14) (J R. Sadler, H. Sasmor and J L. Betz. PNAS. 1983 Nov.; 80(22): 6785-9). A 289 bp HindIII/ SpeI fragment containing the tacpromoter and the native lacO sequence was removed from a pMYC1803derivative, pDOW2118, and replaced by a HindIII/SpeI fragment isolatedfrom an SOE PCR amplification product containing the symmetrical lacOidsequence. The SOE PCR primers (RC-3 and RC-9) incorporated 4 nucleotidechanges that produced the optimized/symmetrical lacO sequence (threebase pair substitutions and one base pair deletion). The HindIII/SpeIpromoter fragment of the resulting plasmid, pDOW2201, was cloned intothe nitrilase expression plasmid based on pMYC1803, to replace thenative tac promoter, resulting in pDOW1414. This expression cassette wasthen transferred onto the pyrF(+) plasmid pDOW1269, resulting inpDOW1418 by exchanging DraI/XhoI fragments. Plasmid pDOW1418 was thentransformed into host strain DC206, resulting in strain DC281 (See FIG.4).

Oligonucleotides Used RC-3 5′-GTGAGCGCTCACAATTCCACACAGGAAA (SEQ ID NO:52) ACAG RC-4 5′-TTCGGGTGGAAGTCCAGGTAGTTGGCGG (SEQ ID NO: 53) TGTA RC-95′-GAATTGTGAGCGCTCACAATTCCACACA (SEQ ID NO: 54) TTATACGAGC RC-105′-ATTCAGCGCATGTTCAACGG (SEQ ID NO: 55)

In the second construct, pDOW1416, the lacOid operator, 5′-AATTGTGAGCGCTCACAATT-3′ (SEQ ID. No. 14), was inserted 52 nucleotides up-stream(5′) of the existing native lacO1 by PCR. PCR amplification of thepromoter region using the Megaprimer method was performed using apMYC1803 derivative, pMYC5088, and the following primers AKB-1 and AKB-2as a first step. The resulting PCR product was combined with primerAKB-3 in a second round of PCR amplification using the same template.After purification and digestion with HindIII and SpeI, the promoterfragment containing the dual operators was cloned into the HindIII andSpeI sites of plasmid pMYC5088 resulting in pDOW1411. Introduction ofthe second operator introduced a unique MfeI site immediately upstreamof the optimal operator. The XhoI/SpeI vector fragment with promoterregions of pDOW1411 was then ligated with the compatible fragment of thepMYC1803 derivative bearing the nitrilase gene, forming pDOW1413.Subsequent ligation of the MfeI/XhoI expression cassette fragment ofpDOW1413 to the compatible vector fragment of pDOW1269 resulted inpDOW1416; which when transformed into DC206, formed the strain DC262.

Oligonucleotides Used AKB-1 5′-ACGGTTCTGGCAAACAATTGTGAGCGCTCAC (SEQ IDNO: 56) AATTTATTCTGAAATGAGC AKB-2 5′-GCGTGGGCGGTGTTTATCATGTTC (SEQ IDNO: 57) AKB-3 5′-TACTGCACGCACAAGCCTGAACA (SEQ ID NO: 58)

Nitrilase Derepression

Northern blot analysis was performed pre and post induction on MB214,DC202, and DC206. MB214, DC202, and DC206 were transformed with anitrilase expression vector containing the wild type lacO sequence inthe O₁ position 3′ of the tac promoter, creating MB214 wtO₁, DC202wtO₁(DC239), and DC206wtO₁ (DC240), as described above. DC206 wastransformed with a nitrilase expression vector containing a lacOidsequence in place of the wild type lacO sequence at the O₁ position 3′of the tac promoter as described above, creating DC206Oid (DC281). DC206was also transformed with a nitrilase expression vector containing awild type lacO sequence at the O₁ position 3′ of the tac promoter and alacOid sequence at the O₃ position 5′ of the tac promoter, creating thedual lacO containing DC206wtO₁ O₃id (DC263).

Northern blot analysis indicated a greater repression by the straincontaining the Dual lacO sequence (DC206wtO₁ O₃id (DC263)) cassetteprior to induction. The greater repression of pre-induction expressionis especially useful when producing toxic proteins, since basal levelsof pre-induction toxic proteins result in the delayed entry of the cellinto the growth phase, and, potentially, lower overall yields of theproduct.

1) An auxotrophic Pseudomonad cell for use in a bacterial expressionsystem that comprises a nucleic acid construct comprising: a. a nucleicacid encoding a recombinant polypeptide; and, b. a nucleic acid encodingat least one polypeptide that restores prototrophy to the auxotrophichost cell. 2) The cell of claim 1, wherein the Pseudomonad isPseudomonas fluorescens. 3) The cell of claim 1, wherein the cell isauxotrophic for uracil. 4) The cell of claim 1, wherein the cell isauxotrophic for proline. 5) The cell of claim 1, wherein the auxotrophiccell is auxotrophic for more than one metabolite. 6) The cell of claim5, wherein the cell is auxotrophic for uracil and proline. 7) The cellof claim 1, wherein the prototrophy restoring polypeptide is an enzymeactive in the biosynthesis of a metabolite required for cell survival.8) The cell of claim 7, wherein the enzyme is orotodine-5′-phosphatedecarboxylase. 9) The cell of claim 8, wherein the enzyme is encoded bythe nucleic acid sequence selected from the group consisting of SEQ. ID.1 and
 3. 10) The cell of claim 7, wherein the enzyme comprises the aminoacid sequence of SEQ ID No.
 2. 11) The cell of claim 7, wherein theenzyme is Δ¹-pyrroline-5-carboxylate reductase. 12) The cell of claim11, wherein the enzyme is encoded by the nucleic acid sequence selectedfrom the group consisting of SEQ. ID. NO. 6 and
 8. 13) The cell of claim11, wherein the enzyme comprises the amino acid sequence of SEQ. ID. No.7. 14) The cell of claim 1, wherein the auxotrophic cell is produced bydisabling a pyrF gene. 15) The cell of claim 14, wherein the disabledpyrF gene comprises the nucleic acid selected from the group consistingof SEQ. ID. No. 1 and SEQ. ID. No.
 3. 16) The cell of claim 1, whereinthe auxotrophic cell is produced by disabling a proC gene. 17) The cellof claim 16, wherein the disabled proC gene comprises the nucleic acidselected from the group consisting of SEQ. ID. No. 6 and SEQ. ID. No. 8.18) The cell of claim 1, wherein the auxotrophic cell is produced bydisabling a pyrF gene and a proC gene. 19) The cell of claim 18, whereinthe disabled pyrF gene comprises the nucleic acid selected from thegroup consisting of SEQ. ID. No. 1 and SEQ. ID. No.3, and the disabledproC gene comprises the nucleic acid selected from the group consistingof SEQ. ID. No. 6 and SEQ. ID. NO.
 9. 20) The cell of claim 1, whereinthe cell also contains a chromosomal lacI insert that is other than aspart of a PlacI-lacI-lacZYA operon. 21) The cell of claim 20, whereinthe lacI gene is selected from the group consisting of lacI, lacI^(Q,)and lacI^(Q1). 22) The cell of claim 1, wherein the nucleic acidconstruct further comprises at least one lacOid sequence. 23) The cellof claim 22, wherein the lacOid sequence is selected from the groupconsisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 24) The cell of claim1, wherein the nucleic acid construct further comprising more than onelac operator sequences. 25) The cell of claim 24, wherein at least onelac operator sequence is located 5′ of a promoter, and at least one lacoperator sequence is located 3′ of a promoter. 26) The cell of claim 25,wherein at least one lac operator sequence is a lacOid sequence. 27) Thecell of claim 26, wherein the lacOid sequence is selected from the groupconsisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 28) A geneticallymodified Pseudomonad cell for use in a bacterial expression system,wherein the modification comprises at least one chromosomal insertion ofa lacI gene, wherein the lacI gene is other than as part of a whole ortruncated PlacI-lacI-lacZYA operon. 29) The cell of claim 28, whereinthe Pseudomonad is Pseudomonas fluorescens. 30) The cell of claim 28,wherein the lacI gene is selected from the group consisting of lacI,lacI^(Q,) and lacI^(Q1.) 31) The cell of claim 28, wherein the lacI geneis inserted in the levansucrase locus. 32) The cell of claim 28, whereinthe cell has been further modified to create an auxotrophy for at leastone metabolite in the cell. 33) The cell of claim 32, wherein theauxotrophy is created by modification to a gene selected from the groupconsisting of pyrF and proC. 34) The cell of claim 32, wherein theauxotrophy is created by modification to both the pyrF and the proCgene. 35) The cell of claim 28, further comprising a nucleic acidcomprising at least one lacOid sequence. 36) The cell of claim 35,wherein the lacOid sequence is selected from the group consisting ofSEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 37) The cell of claim 28 furthercomprising a nucleic acid comprising more than one lac operatorsequence. 38) The cell of claim 37, wherein at least one lac operatorsequence is a lacOid sequence. 39) The cell of claim 38, wherein thelacOid sequence is selected from the group consisting of SEQ. ID. NO. 14and SEQ. ID. NO.
 59. 40) A Pseudomonad cell for use in a bacterialexpression system comprising a nucleic acid construct comprising atleast one lacOid operator sequence. 41) The cell of claim 40, whereinthe Pseudomonad is a Pseudomonas fluorescens. 42) The cell of claim 40,wherein the lacOid sequence is located 3′ of a promoter. 43) The cell ofclaim 40, wherein the lacOid sequence is located 5′ of a promoter. 44)The cell of claim 40, wherein lacOid sequences are located 3′ and 5′ ofa promoter. 45) The cell of claim 40, wherein the cell has been furthermodified to create an auxotrophy for at least one metabolite in thecell. 46) The cell of claim 45, wherein the auxotrophy is created bymodification to a gene selected from the group consisting of pyrF andproC. 47) The cell of claim 45, wherein the auxotrophy is created bymodification to both the pyrF and the proC gene. 48) The cell of claim40, wherein the cell contains a chromosomal insertion of a lacI gene,wherein the lacI gene is other than as part of a whole or truncatedPlac-lacI-lacZYA operon. 49) The cell of claim 40, wherein the lacOidsequence is selected from the group consisting of SEQ ID NO. 14 and SEQ.ID. NO.
 59. 50) A Pseudomonad cell for use in a bacterial expressionsystem comprising a nucleic acid construct comprising more than one lacoperator sequence. 51) The cell of claim 50, wherein at least one lacoperator is a lacOid sequence. 52) The cell of claim 51, wherein thelacOid sequence is selected from the group consisting of SEQ. ID. NO. 14and SEQ. ID. NO.
 59. 53) The cell of claim 51, wherein the lacOidsequence is 5′ or 3′ of the promoter. 54) The cell of claim 51 whereinthe lacOid sequence is 5′ and 3′ of the promoter. 55) The cell of claim50, wherein the cell has been further modified to create an auxotrophyfor at least one metabolite in the cell. 56) The cell of claim 55,wherein the auxotrophy is created by modification to a gene selectedfrom the group consisting of pyrF and proC. 57) The cell of claim 55,wherein the auxotrophy is created by modification to both the pyrF andthe proC gene. 58) The cell of claim 50, wherein the cell contains achromosomal insertion of a lacI gene, wherein the lacI gene is otherthan as part of a whole or truncated Plac-lacI-lacZYA operon. 59) Thecell of claim 50, wherein the Pseudomonad is Pseudomonas fluorescens.60) A process for producing a recombinant polypeptide comprising: a.expressing a nucleic acid encoding the recombinant polypeptide in aPseudomonad cell that has been genetically modified to create anauxotrophy for at least one metabolite; b. expressing a nucleic acidencoding a polypeptide that restores prototrophy to the auxotrophiccell; and, c. growing the cell on a medium that lacks the auxotrophicmetabolite. 61) The process of claim 60, wherein the Pseudomonad isPseudomonas fluorescens. 62) The process of claim 60, wherein the cellis auxotrophic for uracil. 63) The process of claim 60, wherein the cellis auxotrophic for proline. 64) The process of claim 60, wherein theauxotrophic cell is auxotrophic for more than one metabolite. 65) Theprocess of claim 64, wherein the cell is auxotrophic for uracil andproline. 66) The process of claim 60, wherein the prototrophy restoringpolypeptide is an enzyme active in the biosynthesis of a metaboliterequired for cell survival. 67) The process of claim 66, wherein theenzyme is orotodine-5′-phosphate decarboxylase. 68) The process of claim67, wherein the enzyme is encoded by the nucleic acid sequence selectedfrom the group consisting of SEQ. ID. 1 and
 3. 69) The process of claim68, wherein the enzyme comprises the amino acid sequence of SEQ ID No.2. 70) The process of claim 66, wherein the enzyme isΔ¹-pyrroline-5-carboxylate reductase. 71) The process of claim 70,wherein the enzyme is encoded by the nucleic acid sequence selected fromthe group consisting of SEQ. ID. NO. 6 and
 8. 72) The process of claim70, wherein the enzyme comprises the amino acid sequence of SEQ. ID. No.7. 73) The process of claim 60, wherein the auxotrophic cell is producedby disabling a pyrF gene. 74) The process of claim 73, wherein thedisabled pyrF gene comprises the nucleic acid selected from the groupconsisting of SEQ. ID. No. 1 and SEQ. ID. No.
 3. 75) The process ofclaim 60, wherein the auxotrophic cell is produced by disabling a proCgene. 76) The process of claim 75, wherein the disabled proC genecomprises the nucleic acid selected from the group consisting of SEQ.ID. No. 6 and SEQ. ID. No.
 8. 77) The process of claim 60, wherein theauxotrophic cell is produced by disabling a pyrF gene and a proC gene.78) The process of claim 77, wherein the disabled pyrF gene comprisesthe nucleic acid selected from the group consisting of SEQ. ID. No. 1and SEQ. ID. No. 3, and the disabled proC gene comprises the nucleicacid selected from the group consisting of SEQ. ID. No. 6 and SEQ. ID.NO.
 9. 79) The process of claim 60, wherein the cell also contains achromosomal lacI insert that is other than as part of aPlacI-lacI-lacZYA operon. 80) The process of claim 79, wherein the lacIgene is selected from the group consisting of lacI, lacI^(Q,) andlac^(Q1). 81) The process of claim 60, wherein the nucleic acid encodingthe recombinant polypeptide further comprises at least one lacOidsequence. 82) The proves of claim 81, wherein the lacOid sequence isselected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.59. 83) The process of claim 60, wherein the nucleic acid encoding therecombinant polypeptide further comprises more than one lac operatorsequences. 84) The process of claim 83, wherein at least one lacoperator sequence is located 5′ of a promoter, and at least one lacoperator sequence is located 3′ of a promoter. 85) The process of claim84, wherein at least one lac operator sequence is a lacOid sequence. 86)The process of claim 85, wherein the lacOid sequence is selected fromthe group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 87) Aprocess for producing a recombinant polypeptide comprising expressing anucleic acid encoding the recombinant polypeptide in a Pseudomonad thatcomprises at least one chromosomal insertion of a lacI gene, wherein thelacI gene is other than as part of a whole or truncatedPlacI-lacI-lacZYA operon. 88) The process of claim 87, wherein thePseudomonad is Pseudomonas fluorescens. 89) The process of claim 87,wherein the lacI gene is selected from the group consisting of lacI,lacI^(Q,) and lacI^(Q1). 90) The process of claim 87, wherein the lacIgene is inserted in the levansucrase locus. 91) The process of claim 87,wherein the cell has been further modified to create an auxotrophy forat least one metabolite in the cell. 92) The process of claim 91,wherein the auxotrophy is created by modification to a gene selectedfrom the group consisting of pyrF and proC. 93) The process of claim 91,wherein the auxotrophy is created by modification to both the pyrF andthe proC gene. 94) The process of claim 87, wherein the nucleic acidencoding the recombinant polypeptide comprises at least one lacOidsequence. 95) The process of claim 94, wherein the lacOid sequence isselected from the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.59. 96) The process of claim 87, wherein the nucleic acid encoding therecombinant polypeptide comprises more than one lac operator sequence.97) The process of claim 96, wherein at least one lac operator sequenceis a lacOid sequence. 98) The process of claim 97, wherein the lacOidsequence is selected from the group consisting of SEQ. ID. NO. 14 andSEQ. ID. NO.
 59. 99) A process for producing a recombinant polypeptidecomprising expressing a nucleic acid encoding the recombinantpolypeptide in a Pseudomonad cell, wherein the nucleic acid furthercomprises at least one lac operator sequence, wherein the lac operatorsequence is a lacOid sequence. 100) The process of claim 99, wherein thePseudomonad is a Pseudomonas fluorescens. 101) The process of claim 99,wherein the lacOid sequence is selected from the group consisting ofSEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 102) The process of claim 99,wherein at least one lacOid sequence is located 3′ of a promoter. 103)The process of claim 99, wherein at least one lacOid sequence is located5′ of a promoter. 104) The process of claim 99, wherein at least onelacOid sequence is located 3′ and 5′ of a promoter. 105) The process ofclaim 99, wherein the cell has been further modified to create anauxotrophy for at least one metabolite in the cell. 106) The process ofclaim 105, wherein the auxotrophy is created by modification to a geneselected from the group consisting of pyrF and proC. 107) The process ofclaim 105, wherein the auxotrophy is created by modification to both thepyrF and the proC gene. 108) The process of claim 99, wherein the cellcontains a chromosomal insertion of a lacI gene, wherein the lacI geneis other than as part of a whole or truncated Plac-lacI-lacZYA operon.109) A process for producing a recombinant polypeptide comprisingexpressing a nucleic acid encoding the recombinant polypeptide in aPseudomonad cell, wherein the nucleic acid further comprises more thanone lac operator sequence. 110) The process of claim 109, wherein atleast one lac operator is a lacOid sequence. 111) The process of claim110, wherein the lacOid sequence is selected from the group consistingof SEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 112) The process of claim 110,wherein the lacOid sequence is 5′ or 3′ of the promoter. 113) Theprocess of claim 110, wherein the lacOid sequence is 5′ and 3′ of thepromoter. 114) The process of claim 109, wherein the cell has beenfurther modified to create an auxotrophy for at least one metabolite inthe cell. 115) The process of claim 114, wherein the auxotrophy iscreated by modification to a gene selected from the group consisting ofpyrF and proC. 116) The process of claim 114, wherein the auxotrophy iscreated by modification to both the pyrF and the proC gene. 117) Theprocess of claim 109, wherein the cell contains a chromosomal insertionof a lacI gene, wherein the lacI gene is other than as part of a wholeor truncated Plac-lacI-lacZYA operon. 118) The process of claim 109,wherein the Pseudomonad is Pseudomonas fluorescens 119) A process formodulating the expression of a recombinant polypeptide in a host cellcomprising: a. selecting a Pseudomonad cell, wherein the cell has beengenetically modified by chromosomally inserting a lacI gene into thecell, wherein the lacI gene is other than as part of a whole ortruncated PlacI-lacI-lacZYA operon; and, b. introducing into the cell anucleic acid construct comprising a LacI protein promoter operablyattached to a nucleic acid encoding the recombinant polypeptide. 120)The process of claim 119, wherein the Pseudomonad is Pseudomonasfluorescens. 121) The process of claim 119, wherein the lacI gene isselected from the group consisting of lacI, lacI^(Q,) and lacI^(Q1).122) The process of claim 119, wherein the lacI gene is inserted in thelevansucrase locus. 123) The process of claim 119, wherein the cell hasbeen further modified to create an auxotrophy for at least onemetabolite in the cell. 124) The process of claim 123, wherein theauxotrophy is created by modification to a gene selected from the groupconsisting of pyrF and proC. 125) The process of claim 123, wherein theauxotrophy is created by modification to both the pyrF and the proCgene. 126) The process of claim 119, wherein the nucleic acid encodingthe recombinant polypeptide comprises at least one lacOid sequence. 127)The process of claim 126, wherein the lacOid sequence is selected fromthe group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 128) Theprocess of claim 126, wherein at least one lacOid sequence is located 3′of the promoter. 129) The process of claim 126, wherein at least onelacOid sequence is located 5′ of the promoter. 130) The process of claim126, wherein lacOid sequence are located 5′ and 3′ of the promoter. 131)The process of claim 119, wherein the nucleic acid encoding therecombinant polypeptide comprises more than one lac operator sequence.132) The process of claim 131, wherein at least one lac operatorsequence is a lacOid sequence. 133) The process of claim 132, whereinthe lacOid sequence is selected from the group consisting of SEQ. ID.NO. 14 and SEQ. ID. NO.
 59. 134) A process for modulating the expressionof a recombinant polypeptide in a host cell comprising: a. selecting aPseudomonad cell; and b. introducing a nucleic acid constructcomprising: i. a nucleic acid encoding the recombinant polypeptide, and,ii. at least one lacOid operator sequence. 135) The process of claim134, wherein the Pseudomonad is a Pseudomonas fluorescens. 136) Theprocess of claim 134, wherein the lacOid sequence is located 3′ of apromoter. 137) The process of claim 134, wherein the lacOid sequence islocated 5′ of a promoter. 138) The process of claim 134, wherein lacOidsequences are located 3′ and 5′ of a promoter. 139) The process of claim134, wherein the cell has been further modified to create an auxotrophyfor at least one metabolite in the cell. 140) The process of claim 139,wherein the auxotrophy is created by modification to a gene selectedfrom the group consisting of pyrF and proC. 141) The process of claim139, wherein the auxotrophy is created by modification to both the pyrFand the proC gene. 142) The process of claim 134, wherein the cellcontains a chromosomal insertion of a lacI gene, wherein the lacI geneis other than as part of a whole or truncated Plac-lacI-lacZYA operon.143) The process of claim 134, wherein the lacOid sequence is selectedfrom the group consisting of SEQ. ID. NO. 14 and SEQ. ID. NO.
 59. 144) Aprocess for modulating the expression of a recombinant polypeptide in ahost cell comprising: a. selecting a Pseudomonad cell; and b.introducing a nucleic acid construct comprising: i. a nucleic acidencoding the recombinant polypeptide, and, ii. more than one lacoperator sequence. 145) The process of claim 144, wherein at least onelac operator is a lacOid sequence. 146) The process of claim 145,wherein the lacOid sequence is selected from the group consisting ofSEQ. ID. NO.14 and SEQ. ID. NO.
 59. 147) The process of claim 145,wherein the lacOid sequence is 5′ or 3′ of the promoter. 148) Theprocess of claim 145 wherein the lacOid sequence is 5′ and 3′ of thepromoter. 149) The process of claim 144, wherein the cell has beenfurther modified to create an auxotrophy for at least one metabolite inthe cell. 150) The process of claim 149, wherein the auxotrophy iscreated by modification to a gene selected from the group consisting ofpyrF and proC. 151) The process of claim 149, wherein the auxotrophy iscreated by modification to both the pyrF and the proC gene. 152) Theprocess of claim 144, wherein the cell contains a chromosomal insertionof a lacI gene, wherein the lacI gene is other than as part of a wholeor truncated Plac-lacI-lacZYA operon. 153) The process of claim 144,wherein the Pseudomonad is Pseudomonas fluorescens. 154) A process forthe production of a recombinant polypeptide in the absence ofantibiotics comprising: a. selecting a Pseudomonad cell, wherein thecell has been genetically modified to induce an auxotrophy for at leastone metabolite, thereby creating an auxotrophic cell; b. introducinginto the cell a nucleic acid construct comprising i. a nucleic acidencoding the recombinant polypeptide; and ii. a nucleic acid encoding apolypeptide that restores prototrophy to the auxotrophic host cell; c.expressing the recombinant polypeptide and prototrophy restoringpolypeptide in the cell; and, d. growing the cell on a medium that lacksthe auxotrophic metabolite. 155) The process of claim 154, wherein thePseudomonad is Pseudomonas fluorescens. 156) The process of claim 154,wherein the cell is auxotrophic for uracil. 157) The process of claim154, wherein the cell is auxotrophic for proline. 158) The process ofclaim 154, wherein the auxotrophic cell is auxotrophic for more than onemetabolite. 159) The process of claim 158, wherein the cell isauxotrophic for uracil and proline. 160) The process of claim 154,wherein the prototrophy restoring polypeptide is an enzyme active in thebiosynthesis of a metabolite required for cell survival. 161) Theprocess of claim 160, wherein the enzyme is orotodine-5′-phosphatedecarboxylase. 162) The process of claim 161, wherein the enzyme isencoded by the nucleic acid sequence selected from the group consistingof SEQ. ID. 1 and
 3. 163) The process of claim 160, wherein the enzymecomprises the amino acid sequence of SEQ ID No.
 2. 164) The process ofclaim 160, wherein the enzyme is Δ¹-pyrroline-5-carboxylate reductase.165) The process of claim 164, wherein the enzyme is encoded by thenucleic acid sequence selected from the group consisting of SEQ. ID. NO.6 and
 8. 166) The process of claim 164, wherein the enzyme comprises theamino acid sequence of SEQ. ID. No.
 7. 167) The process of claim 154,wherein the auxotrophic cell is produced by disabling a pyrF gene. 168)The process of claim 167, wherein the disabled pyrF gene comprises thenucleic acid selected from the group consisting of SEQ. ID. No.1 andSEQ. ID. No.
 3. 169) The process of claim 154, wherein the auxotrophiccell is produced by disabling a proC gene. 170) The process of claim169, wherein the disabled proC gene comprises the nucleic acid selectedfrom the group consisting of SEQ. ID. No. 6 and SEQ. ID. No.
 8. 171) Theprocess of claim 154, wherein the auxotrophic cell is produced bydisabling a pyrF gene and a proC gene. 172) The process of claim 171,wherein the disabled pyrF gene comprises the nucleic acid selected fromthe group consisting of SEQ. ID. No.1 and SEQ. ID. No. 3, and thedisabled proC gene comprises the nucleic acid selected from the groupconsisting of SEQ. ID. No. 6 and SEQ. ID. NO.9. 173) The process ofclaim 154, wherein the cell also contains a chromosomal lacI insert thatis other than as part of a PlacI-lacI-lacZYA operon. 174) The process ofclaim 173, wherein the lacI gene is selected from the group consistingof lacI, lacI^(Q,) and lacI^(Q1). 175) The process of claim 154, whereinthe nucleic acid construct further comprises at least one lacOidsequence. 176) The process of claim 154, wherein the nucleic acidconstruct further comprising more than one lac operator sequences. 177)The process of claim 176, wherein at least one lac operator sequence islocated 5′ of a promoter, and at least one lac operator sequence islocated 3′ of a promoter. 178) The process of claim 177, wherein atleast one lac operator sequence is a lacOid sequence. 179) A process forthe production of a recombinant polypeptide in the absence ofantibiotics wherein cross feeding inhibition is minimized duringselection comprising: a. selecting a Pseudomonad cell, wherein the cellhas been genetically modified to induce an auxotrophy for at least onemetabolite, thereby creating an auxotrophic cell; b. introducing intothe cell a nucleic acid construct comprising i. a nucleic acid encodinga recombinant polypeptide; and ii. a nucleic acid encoding a polypeptidethat restores prototrophy to the auxotrophic host cell; c. expressingthe recombinant polypeptide and the prototrophy restoring polypeptide inthe cell; and, d. growing the cell on a medium that lacks theauxotrophic metabolite. 180) The process of claim 179, wherein thePseudomonad is Pseudomonas fluorescens. 181) The process of claim 179,wherein the cell is auxotrophic for uracil. 182) The process of claim179, wherein the cell is auxotrophic for proline. 183) The process ofclaim 179, wherein the auxotrophic cell is auxotrophic for more than onemetabolite. 184) The process of claim 183, wherein the cell isauxotrophic for uracil and proline. 185) The process of claim 179,wherein the prototrophy restoring polypeptide is an enzyme active in thebiosynthesis of a metabolite required for cell survival. 186) Theprocess of claim 185, wherein the enzyme is orotodine-5′-phosphatedecarboxylase. 187) The process of claim 186, wherein the enzyme isencoded by the nucleic acid sequence selected from the group consistingof SEQ. ID. 1 and
 3. 188) The process of claim 185, wherein the enzymecomprises the amino acid sequence of SEQ ID No.
 2. 189) The process ofclaim 185, wherein the enzyme is Δ¹-pyrroline-5-carboxylate reductase.190) The process of claim 189, wherein the enzyme is encoded by thenucleic acid sequence selected from the group consisting of SEQ. ID. NO.6 and
 8. 191) The process of claim 189, wherein the enzyme comprises theamino acid sequence of SEQ. ID. No.
 7. 192) The process of claim 179,wherein the auxotrophic cell is produced by disabling a pyrF gene. 193)The process of claim 192, wherein the disabled pyrF gene comprises thenucleic acid selected from the group consisting of SEQ. ID. No. 1 andSEQ. ID. No.
 3. 194) The process of claim 179, wherein the auxotrophiccell is produced by disabling a proC gene. 195) The process of claim194, wherein the disabled proC gene comprises the nucleic acid selectedfrom the group consisting of SEQ. ID. No. 6 and SEQ. ID. No.
 8. 196) Theprocess of claim 179, wherein the auxotrophic cell is produced bydisabling a pyrF gene and a proC gene. 197) The process of claim 196,wherein the disabled pyrF gene comprises the nucleic acid selected fromthe group consisting of SEQ. ID. No. 1 and SEQ. ID. No.3, and thedisabled proC gene comprises the nucleic acid selected from the groupconsisting of SEQ. ID. No. 6 and SEQ. ID. NO.
 9. 198) The process ofclaim 179, wherein the cell also contains a chromosomal lacI insert thatis other than as part of a PlacI-lacI-lacZYA operon. 199) The process ofclaim 198, wherein the lacI gene is selected from the group consistingof lacI, lacI^(Q,) and lacI^(Q1). 200) The process of claim 199, whereinthe nucleic acid construct further comprises at least one lacOidsequence. 201) The process of claim 179, wherein the nucleic acidconstruct further comprising more than one lac operator sequences. 202)The process of claim 201, wherein at least one lac operator sequence islocated 5′ of a promoter, and at least one lac operator sequence islocated 3′ of a promoter. 203) The process of claim 202, wherein atleast one lac operator sequence is a lacOid sequence. 204) A Pseudomonasfluorescens pyrF gene, or nucleic acid that hybridizes with the pyrFgene, comprising the nucleic acid sequence selected from the groupconsisting of SEQ. ID. No. 1 or
 3. 205) The gene of claim 204, whereinthe nucleic acid comprises the sequence of SEQ. ID. No.
 1. 206) The geneof claim 204, wherein the nucleic acid comprises the sequence of SEQ.ID. No. 3 207) A Pseudomonas fluorescens proC gene, or nucleic acid thathybridizes with the proC gene, comprising the nucleic acid sequenceselected from the group consisting of SEQ. ID. No. 6 or
 8. 208) The geneof claim 207, wherein the nucleic acid comprises the sequence of SEQ IDNo.
 6. 209) The gene of claim 207, wherein the nucleic acid comprisesthe sequence of SEQ ID No.
 8. 210) A nucleic acid construct comprising:a. a nucleic acid encoding a recombinant polypeptide; and b. a nucleicacid encoding a pyrF gene isolated from a Pseudomonas fluorescens. 211)The construct of claim 210, wherein the pyrF gene comprises the nucleicacid sequence of SEQ. ID No.
 1. 212) The construct of claim 210, whereinthe pyrF gene comprises the nucleic acid sequence of SEQ. ID No.
 3. 213)A nucleic acid construct comprising: a. a nucleic acid encoding arecombinant polypeptide; and b. a nucleic acid encoding a proC geneisolated from a Pseudomonas fluorescens. 214) The construct of claim213, wherein the proC gene comprises the nucleic acid sequence of SEQ IDNo.
 6. 215) The construct of claim 213, wherein the proC gene comprisesthe nucleic acid sequence of SEQ ID No. 8.